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Endocrinology Vol. 141, No. 10 3556-3563
Copyright © 2000 by The Endocrine Society

ARTICLES

Modulation of Pituitary Somatostatin Receptor Subtype (sst1–5) Messenger Ribonucleic Acid Levels by Changes in the Growth Hormone Axis1

Seungjoon Park2, Jun Kamegai3, Todd A. Johnson4, Lawrence A. Frohman and Rhonda D. Kineman

Department of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Rhonda D. Kineman, Ph.D., Department of Medicine (M/C 640), University of Illinois at Chicago, 1819 West Polk, Chicago, Illinois 60612. E-mail: kineman{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of individual components of the hypothalamic-pituitary-GH axis in the modulation of pituitary somatostatin (SRIF) receptor subtype (sst1–5) synthesis was assessed using multiplex RT-PCR to measure receptor messenger RNA (mRNA) levels in normal rats and spontaneous dwarf rats (SDRs). In SDRs, a strain with no immunodetectable GH, pituitary sst1 and sst2 mRNA levels were elevated, sst5 mRNA levels were reduced, and sst3 and sst4 mRNA levels did not significantly differ from those in normal controls. Treatment of SDRs with GH (72 h), but not insulin-like growth factor I, significantly decreased sst2 mRNA levels and increased sst4 and sst5 mRNA levels above vehicle-treated control levels. To test whether more rapid changes in circulating GH levels could alter SRIF receptor subtype expression, normal rats were infused (iv) with GH-releasing hormone (GHRH) for 4 h in the presence or absence of SRIF antiserum. GHRH infusion increased pituitary sst1 and sst2 and decreased sst5, but had no effect on sst3 and sst4 mRNA levels. Immunoneutralization of SRIF, which produced a rise in circulating GH levels, did not alter basal or GHRH-mediated SRIF receptor subtype expression. These observations indicate that acute suppression of SRIF tone does not regulate pituitary SRIF receptor subtype mRNA levels in vivo. The possibility that elevated circulating GH concentrations induced by GHRH infusion were responsible for the observed changes in SRIF receptor subtype mRNA levels was examined by infusing SDRs with GHRH for 4 h. GHRH did not increase sst1 mRNA levels in SDRs above their already elevated value. However, GHRH infusion produced an increase in sst2 and a decrease in sst5 mRNA levels similar to those observed in normal rats, indicating that the acute effects of GHRH on SRIF receptor subtype expression are independent of circulating GH levels. Primary rat pituitary cell cultures were incubated with GHRH (10 nM) or forskolin (10 µM) for 4 h to determine whether GHRH could directly mediate SRIF receptor subtype mRNA. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst5 mRNA levels, but had no effect on sst3 and sst4, similar to the results in vivo. The effect of forskolin mimicked that of GHRH on sst1, sst2, and sst5 mRNA, suggesting that GHRH acts through cAMP to directly mediate gene transcription or mRNA stability of these SRIF receptor subtypes. In addition, forskolin reduced sst3 and sst4 expression. These results strongly suggest that rat pituitary sst1, sst2, and sst5 mRNA levels are regulated both in vivo and in vitro by GHRH. The stimulatory action of GHRH on sst1 and sst2 and the inhibitory action on sst5 indicate that these receptor subtypes have independent and unique roles in the modulation of pituitary GH release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN (SRIF) is widely distributed throughout the central nervous system and peripheral tissues and exerts a variety of physiological actions, including the inhibition of GH release from anterior pituitary somatotropes (1, 2, 3). The actions of SRIF are mediated through specific membrane-bound, high affinity, G protein-coupled receptors. SRIF receptors are encoded by five separate genes (sst1–5) (4, 5). All sst subtypes are expressed in the rat anterior pituitary; however, in situ hybridization and immunocytochemistry studies indicate that SRIF receptor expression patterns are cell type specific (6, 7, 8, 9). The relative number of somatotropes that express each SRIF receptor subtype in descending order is sst5 > sst2 > sst3 = sst4 > sst1. sst2 and sst5 also appear to be the dominant subtypes in the human somatotrope, as GH-secreting adenomas demonstrate higher expression levels of sst2 and sst5 messenger RNA (mRNA) than pituitary adenomas that do not express GH or than normal pituitaries (10, 11, 12).

Circulating GH exerts a negative feedback effect at both the hypothalamic and pituitary levels to suppress further GH production. Specifically, an increase in circulating GH leads to a decrease in hypothalamic GH-releasing hormone (GHRH) (13, 14, 15) and an increase in SRIF (15, 16, 17, 18) and neuropeptide Y (NPY) (19), whereas the expression of the anterior pituitary receptors for GHRH (20) and the GH secretagogues (GHS) (21) is suppressed. Collectively, these observations suggest that multiple components of the GH axis work in a coordinate fashion to maintain circulating GH levels in a well defined range. Therefore, it can be reasoned that modulation of pituitary SRIF receptor expression might also be an important component of the GH negative feedback system. However, unlike the GH stimulatory receptors (GHRH-R and GHS-R), it might be expected that the synthesis of pituitary SRIF receptor subtypes would be elevated in the face of increased GH concentrations. This hypothesis is indirectly supported by the fact that sst1, sst2, and sst3 mRNA levels are decreased in the fasted rat, and sst1, sst2, sst3, and sst5 mRNA levels are decreased in the streptozotocin-induced diabetic rat (22), both of which are characterized by low circulating GH levels.

To directly test whether changes in circulating GH are associated with changes in SRIF receptor synthesis, we compared the expression pattern of SRIF receptor subtypes in anterior pituitaries of GH-deficient spontaneous dwarf rats (SDRs) (23) with or without 72-h GH or insulin-like growth factor I (IGF-I) replacement therapy. To determine whether more acute changes in the components of the GH axis could alter the expression of SRIF receptors, we also examined pituitary SRIF receptor expression after 4-h GHRH infusion in the presence or absence of SRIF antiserum and endogenous GH production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experiments
Total RNA was isolated from whole rat pituitaries or primary rat pituitary cell cultures as previously described (20, 21, 24). Pituitary SRIF receptor subtype mRNA levels were determined by RT-PCR (see below for details) in the following experimental groups: Exp 1, normal Sprague Dawley rats (300–400 g) and age-matched SDRs (85–105 g); Exp 2, SDRs infused with rat GH (10 µg/µl; Dr. A. F. Parlow, National Hormone and Pituitary Program, Torrance, CA), recombinant human IGF-I (5 µg/µl; Genentech, Inc., South San Francisco, CA), or vehicle via osmotic minipumps at a rate of 1 µl/h for 72 h; Exp 3, normal Sprague Dawley rats or SDRs infused for 4 h iv with vehicle, a GHRH analog [(des-NH2Tyr1,D-Ala2, Ala15)human GRF-(1–29)-NH2; Dr. R. M. Campbell, Hoffmann La Roche, Inc., Nutley, NJ; 10 µg/h], or a nonpeptidyl GHS (L692,585; 100 µg/h; Dr. R. G. Smith, Merck & Co., Rahway, NJ); a subset of normal rats was also injected iv with SRIF antiserum (0.5 ml/rat; Dr. A. Arimura, Tulane University, New Orleans, LA) 5 min before the initiation of vehicle or GHRH infusion; and Exp 4, primary rat pituitary cell cultures treated for 4 h with GHRH (10 nM) or forskolin (10 µM). All experiments were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals and all protocols were approved by the University of Illinois (Chicago, IL) animal care committee.

Multiplex RT-PCR of SRIF receptor subtypes
One microgram of total pituitary RNA was reverse transcribed using the Superscript Preamplification System for First Strand Synthesis (Life Technologies, Inc., St. Louis, MO) with random hexamer priming. A RNA control tube containing all RT reagents except reverse transcriptase was included to monitor genomic DNA contamination. The resultant complementary DNA (cDNA) from RNA extracts of whole pituitaries was amplified in two separate PCR reactions containing primers for rat sst2 (0.6 µM), sst5 (0.6 µM) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 0.07 µM; as a control) or primers for sst1 (0.4 µM), sst3 (0.2 µM), sst4 (0.6 µM), and GAPDH (0.035 µM). As SRIF receptor mRNA levels in vitro were 5- to 10-fold lower than receptor mRNA levels in whole pituitary extracts, primer concentrations used in the PCR of the in vitro samples were modified to favor amplification (reaction 1: sst2, 0.6 µM; sst5, 0.6 µM; and GAPDH, 0.035 µM; reaction 2: sst1, 0.6 µM; sst3, 0.4 µM; sst4, 0.6 µM; and GAPDH, 0.035 µM). PCR primer sequences for each of the rat sst subtypes and GAPDH were as follows: sst1: sense, 5'-CTA CTT TGC CGC CTG GTG CTC-3'; and antisense, 5'-TGG CAA TGA TGA GCA CGT AAC-3' [GenBank Accession No. (ACC) X62314]; sst2: sense, 5'-TTG ACG GTC ATG AGC ATC G-3'; and antisense, 5'-ACA GAC ACG GAC GAG ACA TTG-3' (ACC no. M93273); sst3:sense, 5'-GGC CGC TGT TAC CTA TCC TTC-3'; and antisense, 5'-GGC ACT CCT GAG AAC ACA ACC-3' (ACC no. X63574); sst4: sense, 5'-CGG AGA CGC TCA GAG AAG AAG-3'; and antisense, 5'-TGG TCT TGG TGA AAG GGA CTC-3' (ACC no. M96544); sst5: sense, 5'-CAT GAG TGT TGA CCG CTA CC-3'; and antisense, 5'-GGC ACA GCT ATT GGC ATA AG-3' (ACC no. L04535); and GAPDH: sense, 5'-AGG GCT GCC TTC TCT TGT GAC A-3'; and antisense, 5'-CAG CAT CAA AGG TGG AAG AAT-3' (ACC no. X02231). Primer sequences were selected that differed by no more than 1.0 C in annealing temperature. The expected sizes of PCR products were 364 bp for sst1, 449 bp for sst2, 555 bp for sst3, 409 bp for sst4, 508 bp for sst5, and 835 bp for GAPDH. In that the relative level of mRNA for each transcript varies greatly for the individual SRIF receptor subtypes and GAPDH, the appropriate primer concentrations were empirically determined to achieve a final signal that was comparable for all PCR products within each reaction and that would provide noncompetitive and specific amplification for each PCR product. Therefore, this technique can only be used to compare individual SRIF receptor subtype expression levels between experimental groups and not the relative expression levels between SRIF receptor subtypes. All PCR reactions were performed in a 50-µl volume containing 2 µl RT reaction, 1 x PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxy-NTPs, 2 U Taq Gold polymerase (Perkin-Elmer Corp., Branchburg, NJ), and 5 µCi [{alpha}-32P]deoxy-CTP (SA, 800 Ci/mmol). The thermal cycling profile was as follows: 95 C for 10 min, 24 (sst2, sst5, GAPDH) or 28 (sst1, sst3, sst4, GAPDH) cycles of 95 C for 30 s, 60 C for 1 min, and 72 C for 1 min. The final extension was at 72 C for 10 min. Reaction products were separated on 5% polyacrylamide-8 M urea gels. Gels were dried on chromatography paper and exposed to a phosphorimage screen.

Statistical analysis
Background levels taken from each lane were subtracted from each specific band signal within that lane. The background-corrected signals for the SRIF receptor PCR products were then adjusted by the background-corrected GAPDH signal, and the data were expressed as a percentage of the control or vehicle-treated value, which was set at 100%. GAPDH was considered an appropriate control, in that GAPDH expression levels did not significantly differ between groups within each experiment. Differences in pituitary expression of SRIF receptor subtypes between normal and SDR animals, the effects of GH and IGF-I treatment in SDRs, and the in vitro effects of GHRH and forskolin were determined by two-tailed Student’s t test. The effects of GHRH on pituitary receptor mRNA levels in the absence or presence of SRIF antiserum were evaluated by two-way ANOVA, and differences between treatment means were determined by Duncan’s new multiple range test. P < 0.05 was considered significant. All comparisons were made between samples electrophoresed on the same gel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validation of the multiplex RT-PCR for pituitary SRIF receptor subtype mRNA levels
The multiplex RT-PCR assay used in this study amplified sst2, sst5, and GAPDH or sst1, sst3, sst4, and GAPDH transcripts from whole pituitary extracts in a single tube, resulting in PCR products of the expected sizes (Fig. 1Go, A and B). There was no amplified product from the negative RT control (data not shown). To determine the cycle numbers that would correspond to the parallel amplification range of all PCR products within each reaction, PCR was performed between 19–37 cycles on cDNA generated from a single RT reaction. All transcripts were amplified with similar efficiencies between 19–27 cycles for sst2, sst5, and GAPDH (Fig. 1AGo) and between 20–28 cycles for sst1, sst3, sst4, and GAPDH (Fig. 1BGo); therefore, all subsequent PCRs were performed at 24 and 28 cycles, respectively. Similar results were obtained in 2 independent experiments, indicating that 1) there is no competition between primer sets; 2) reagents are not limiting; and 3) amplification efficiencies are similar for all transcripts within each reaction. Validation experiments were also performed using RNA extracts from primary rat pituitary cell cultures with similar results (data not shown).



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  		Figure 1. Amplification kinetics of pituitary SRIF receptor subtypes (sst1–5) and GAPDH cDNA by multiplex RT-PCR. One microgram of total pituitary RNA was reverse transcribed using random hexamer priming. cDNA was amplified by PCR in a single sample in the presence of radiolabeled [{alpha}-32P]deoxy-CTP using specific primers for rat sst2, sst5, and GAPDH (A) or sst1, sst3, sst4, and GAPDH (B). Refer to text for specific primer sequences. The radiolabeled PCR products were separated on a 5% polyacrylamide-8 M urea gel. Gels were dried on chromatography paper and exposed to a phosphorimage screen. The signal intensities of the PCR products (A and B, upper panels) were measured by phosphorimager, and pixel density was quantified by image analysis software. All PCR products were of the expected size. Amplifications of sst2, sst5, and GAPDH PCR products were parallel between 19 and 27 cycles (A, lower panel), whereas amplifications of sst1, sst3, sst4, and GAPDH were parallel between 20–28 cycles. Similar results were obtained in 2 separate experiments. Therefore, all subsequent PCR amplifications were performed at 24 cycles for sst2, sst5, and GAPDH and at 28 cycles for sst1, sst3, sst4, and GAPDH. It should be noted that the gel shown in B was computer enhanced and exposed for a longer period of time than the gel shown in A so as to visualize sst4.

 
Pituitary SRIF receptor subtype mRNA levels in normal rats and SDRs
Figure 2Go compares pituitary SRIF receptor subtype mRNA levels between normal rats and SDRs. Pituitary sst1 and sst2 mRNA levels of SDRs were increased to 135% (P < 0.05) and 166% (P < 0.01) of normal control levels, respectively, whereas sst5 mRNA levels were reduced to 42% (P < 0.01). sst3 and sst4 mRNA levels of SDRs were not statistically different from those of normal control values, although there was a trend for sst4 mRNA levels to be reduced in SDRs. The variability observed in sst4 mRNA levels may be related to its low level of expression (Fig. 2BGo).



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  		Figure 2. Multiplex RT-PCR analysis of pituitary SRIF receptor subtype (sst1–5) mRNA levels of SDRs and normal controls. The top panel provides representative examples of phosphorimages obtained after PCR amplification of sst2, sst5, and GAPDH (left) and sst1, sst3, sst4, and GAPDH (right) from normal (N) and SDR (S) pituitary cDNA. The bottom panel illustrates the relative receptor mRNA levels for each SRIF receptor subtype. Signals for each SRIF receptor product were adjusted by GAPDH and expressed as a percentage of normal values. Values represent the mean ± SEM (normal, n = 5; SDR, n = 4). *, P < 0.05; **, P < 0.01.

 
Effect of GH and IGF-I treatment on pituitary SRIF receptor subtype mRNA levels in SDRs
Seventy-two-hour GH replacement therapy, which raised serum IGF-I to normal levels (20), decreased sst2 mRNA to 62% of the vehicle-treated control value (P < 0.05). Conversely, GH infusion increased sst4 and sst5 mRNA levels to 295% (P < 0.01) and 213% (P < 0.01) of control values, respectively (Fig. 3Go). However, sst1 and sst3 mRNA levels were not changed by GH treatment. In contrast to the effects of GH, IGF-I infusion did not significantly alter SRIF receptor subtype mRNA (Fig. 3Go).



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  		Figure 3. Effects of GH (A) and IGF-I (B) treatment on SDR pituitary SRIF receptor subtype (sst1–5) mRNA levels. SDRs were infused for 3 days with vehicle (saline), rat GH (10 µg/h), or recombinant human IGF-I (5 µg/h) via osmotic minipumps. Pituitary sst1–5 mRNA levels were determined by multiplex RT-PCR. Receptor mRNA levels were adjusted by GAPDH and expressed as percentage of vehicle-treated control levels. Values represent the mean ± SEM (n = 5 animals/group). *, P < 0.05; **, P < 0.01.

 
Effect of GHRH infusion on pituitary SRIF receptor subtype mRNA levels
Figure 4Go shows the effect of a 4-h GHRH infusion on SRIF receptor subtype mRNA levels in normal rat pituitaries in the presence or absence of SRIF antiserum. GHRH infusion, which increased circulating GH levels 10-fold (24), also increased sst1 and sst2 mRNA levels to 154% (P < 0.05) and 190% (P < 0.01) of saline-treated control levels, respectively, whereas sst5 mRNA levels were decreased by 56% (P < 0.01). Immunoneutralization of endogenous SRIF did not affect SRIF receptor subtype mRNA levels in the absence or presence of GHRH. The effects of GHRH infusion on pituitary sst2 and sst5 mRNA levels in the SDRs (Fig. 5Go) were similar to those observed in normal rats (Fig. 4Go). In the absence of endogenous GH, GHRH increased sst2 mRNA levels to 196% (P < 0.01) and decreased sst5 levels to 45% (P < 0.01) of vehicle-treated controls. However, GHRH infusion did not significantly affect sst1, sst3, and sst4 mRNA levels in SDRs. In addition, a 4-h infusion of the synthetic GH secretagogue, L692,585, did not significantly affect the level of any SRIF receptor transcript (data not shown).



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  		Figure 4. Effect of GHRH infusion on pituitary SRIF receptor subtype (sst1–5) mRNA levels in the presence (A) and absence (B) of endogenous SRIF. Normal male Sprague Dawley rats were infused for 4 h with either a GHRH analog (10 µg/h) or vehicle. Five minutes before the start of the GHRH infusion, half of the animals received an iv injection (0.5 ml/rat) of SRIF antiserum (ASS) or normal sheep serum (NSS). Pituitary sst1–5 mRNA levels were determined by multiplex RT-PCR. Relative receptor mRNA levels were adjusted by GAPDH and expressed as a percentage of vehicle-treated control levels. Values represent the mean ± SEM (n = 5 animals/group) *, P < 0.05; **, P < 0.01.

 


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  		Figure 5. Effect of GHRH infusion on pituitary SRIF receptor subtype (sst1–5) mRNA levels in male SDRs. Animals were infused for 4 h with either a GHRH analog (10 µg/h) or vehicle, and sst1–5 mRNA levels were determined by multiplex RT-PCR. Relative SRIF receptor subtype mRNA levels were adjusted by GAPDH. Results are expressed as a percentage of vehicle-treated control levels and represent the mean ± SEM (n = 5 animals/group). **, P < 0.01.

 
In vitro effects of GHRH and forskolin on pituitary SRIF receptor subtype mRNA levels
The in vitro effects of GHRH and forskolin on SRIF receptor subtype mRNA levels in primary pituitary cell cultures are presented in Fig. 6Go. GHRH (10 nM) increased sst1 and sst2 mRNA levels to 171% (P < 0.05) and 133% (P < 0.01) of vehicle-treated control values, respectively. In contrast, GHRH decreased sst5 mRNA levels to 47% of control levels (P < 0.01). sst3 and sst4 mRNA levels were not significantly altered by acute GHRH treatment. Forskolin (10 µM) increased sst1 and sst2 mRNA levels to 175% (P < 0.05) and 144% (P < 0.05) of vehicle-treated control values, respectively, whereas forskolin decreased sst3, sst4, and sst5 mRNA levels to 26%, 61%, and 39% of controls (P < 0.01).



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  		Figure 6. Effects of GHRH (A) and forskolin (B) in vitro on pituitary SRIF receptor subtype (sst1–5) mRNA levels. Normal rat pituitaries were enzymatically dispersed and plated at 1 x 106 cells/well in {alpha}MEM/10% horse serum. After 3 days of culture, cells were washed in serum-free medium and incubated with GHRH (10 nM) or forskolin (10 µM) for 4 h, and SRIF receptor subtype mRNA levels were determined by multiplex RT-PCR. sst1-5 mRNA levels were adjusted by GAPDH and expressed as a percentage of vehicle-treated control levels. Values represent the mean ± SEM (n = 4 wells/treatment group). *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study demonstrate pituitary expression of the various SRIF receptor subtypes do not necessarily correlate with circulating GH. In this report, pituitary sst1 and sst2 mRNA levels of the SDR were actually elevated in the absence of GH, whereas sst5 mRNA levels were reduced compared with normal control values. In contrast, experiments by Bruno and co-workers (22) demonstrated a positive correlation between circulating GH levels and pituitary SRIF receptor expression. In their report, pituitary sst1, sst2, sst3, and sst5 mRNA levels were suppressed in the streptozotocin-induced diabetic rat, an animal model with low circulating GH concentrations. Insulin replacement, which restored GH levels (25, 26), partially or completely restored sst1 and sst5 expression, respectively. In addition, they observed that sst1, sst2, and sst3 mRNA levels were reduced in the fasted rat, which is also characterized by low serum GH levels (27). The divergent results for the SDR, fasted, and streptozotocin-treated rat demonstrate that the level of circulating GH does not always directly reflect pituitary SRIF receptor expression and suggest that other factors are involved. Although circulating GH levels are suppressed or absent in each of the animal models studied, the sequence of events that leads to GH deficiency differs. In the SDR, GH secretion and circulating GH are undetectable due to a point mutation in the GH gene, resulting in a truncated and unstable GH mRNA (23). The lack of GH negative feedback in this model, similar to that observed in the hypophysectomized rat (13), leads to an increase in GHRH mRNA and a decrease in SRIF and NPY mRNA levels (20). In contrast, the reduced levels of GH in the fasted rat are thought to be due to a decline in circulating leptin levels, which specifically modulates hypothalamic function (28, 29). The fall in leptin stimulates the activity of hypothalamic NPY neurons (28), leading to an increase in portal levels of SRIF (30) and a decrease in GHRH mRNA levels (31). Likewise, in the streptozotocin-induced diabetic rat, serum leptin levels are reduced due to metabolic changes brought about by hypoinsulinemia. In this model, as in the fasted rat, hypothalamic GHRH mRNA levels are reduced (32), and NPY mRNA levels are increased (33, 34) along with a rise in portal SRIF concentrations (26). A unifying constant in these three models of GH deficiency is the fact that sst1 and sst2 mRNA levels are positively correlated with hypothalamic GHRH expression and negatively correlated with NPY mRNA levels and SRIF tone (summarized in Table 1Go, in vivo experiments). It is difficult to determine from these studies whether any of these neuropeptides are directly responsible for the observed changes in pituitary SRIF receptor expression in the SDR, fasted rat, or streptozotocin-diabetic rat. In each of these paradigms, genetic and experimentally induced alterations in the hypothalamic-pituitary-GH axis occur over a period of days or months, allowing ample time for downstream activation or suppression of additional factors that could be critical for pituitary SRIF receptor regulation.


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  		Table 1. Relationship among circulating hormones, hypothalamic input, and pituitary sst1-5 mRNA levels in the rat

 
To determine whether more rapid changes in the GH axis could affect pituitary SRIF receptor subtype mRNA levels, we used pituitary mRNA samples initially generated to examine the acute regulatory actions of GHRH on its own receptor synthesis in vivo (24). In these samples, a 4-h infusion of GHRH increased pituitary sst1 and sst2 mRNA levels, but decreased sst5 mRNA levels. However, GHRH did not affect sst3 and sst4 expression. As GHRH infusion also caused a dramatic increase in circulating GH levels, which could feed-back at the level of the hypothalamus and increase SRIF tone (20, 35, 36), another group of animals was pretreated with SRIF antiserum. Immunoneutralization of SRIF produced a rise in circulating GH concentrations (24), but did not alter basal or GHRH-mediated SRIF receptor subtype expression. These results indicate that acute changes in SRIF tone do not regulate pituitary SRIF receptor subtype mRNA levels in vivo. This is in contrast to the observation that SRIF can regulate the production of its own receptors in the rat pituitary GH3 cell line (37). Specifically, SRIF was shown to have a biphasic effect on sst2 mRNA levels, which increased at 2 h and decreased at 6 h after addition of the ligand, whereas sst1, sst3, sst4, and sst5 mRNA levels increased at 24 and 48 h. Three possibilities could explain the discrepancy between our in vivo data and those generated using the GH3 cell line. First, the present study examined the effect of suppressing the actions of endogenous SRIF, whereas studies conducted using GH3 cells examined the effect of exogenous SRIF stimulation (37). Second, compensatory mechanisms could occur in vivo to mask the ligand-mediated regulation of SRIF receptor mRNA observed in vitro. Finally, regulatory mechanisms important in SRIF receptor expression may be altered in the immortalized GH3 cell line and therefore may not be representative of SRIF receptor regulation in normal pituitary cells.

To test whether the massive increase in circulating GH levels induced by GHRH infusion could be directly or indirectly responsible for the observed alterations in SRIF receptor subtype mRNA levels, SDRs were infused with GHRH. GHRH infusion increased SDR sst2 and decreased sst5 expression, similar to the effects observed in normal rats. These results indicate that the acute action of GHRH on sst2 and sst5 are GH independent and suggest that GHRH may have a direct effect on pituitary sst2 and sst5 expression. In contrast to the stimulatory effect of GHRH infusion in normal rats, sst1 mRNA levels were not altered by acute GHRH treatment in SDRs. Two possibilities could account for this lack of response: 1) GH is required for GHRH-mediated stimulation of sst1; or 2) sst1 mRNA levels are already up-regulated in the SDR pituitary and therefore are not responsive to additional stimulation. The latter possibility appears to be more likely, in that GHRH increased sst1 as well as sst2 and decreased sst5 mRNA levels in primary rat pituitary cell cultures. As GHRH receptors have only been shown to be expressed in the anterior pituitary somatotrope population (38) the GHRH-mediated changes in sst1, sst2, and sst5 mRNA levels observed in vitro can be attributed to alterations in SRIF receptor gene transcription or SRIF receptor mRNA stability selectively within GH-producing cells. These direct actions of GHRH on SRIF receptor expression may be mediated by activation of the cAMP intracellular signal transduction pathway, as forskolin, a receptor-independent stimulator of adenylyl cyclase, significantly up-regulated sst1 and sst2 and down-regulated sst5 expression in vitro comparable to the effects observed with GHRH. In addition, forskolin suppressed sst3 and sst4 mRNA levels. The ability of forskolin to modulate all SRIF receptor subtypes coupled with the fact that GHRH failed to alter sst3 and sst4 mRNA levels in vitro suggest that the forskolin-mediated fall in sst3 and sst4 expression occurs in pituitary cell types other than somatotropes. Of all of the SRIF receptor subtypes, only the mouse sst2 gene promoter has been shown to contain consensus cAMP response elements (39, 40). Functional studies support a direct action of cAMP on mouse sst2 gene transcription, in that forskolin increased sst2 mRNA levels in the mouse ACTH-producing tumor cell line, AtT-20 (41). The mechanism by which cAMP acts to positively modulate sst1 and negatively modulate sst3, sst4, and sst5 pituitary expression remains to be determined.

In all of the experimental paradigms examined in this report, changes in endogenous and exogenous GHRH were positively correlated with pituitary sst1 and sst2 and negatively correlated with sst5 expression (summarized in Table 1Go). The physiological relevance of these observations may be related to the fact that the specific activation of sst1, sst2, and sst5 leads to inhibition of GH release. It has been reported that SRIF receptor-selective analogs for sst1/5 (42, 43, 44) and sst2 (42, 44), but not sst3 and sst4 (42), suppress basal and cAMP-stimulated GH release from rat and mouse primary pituitary cell cultures. The significance of sst1 in mediating SRIF-induced suppression of GH release was recently confirmed using SRIF receptor-specific knockout mice in which deletion of the sst1 gene blocked the ability of a selective sst1 analog to suppress GH release in primary pituitary cell cultures (43). Therefore, there is strong pharmacological evidence that sst1, sst2, and sst5 are all mediators of SRIF-induced suppression of pituitary GH release. The expression patterns of the various SRIF receptor subtypes within the pituitary cell populations support a dominant role for sst2 and sst5 in regulation of somatotrope function. More somatotropes express sst2 and sst5 mRNA than any other SRIF receptor subtypes, whereas sst1 is expressed in only a small fraction of GH-producing cells (6, 8, 9).

Our results coupled with the functional role of sst1, sst2, and sst5 in mediating GH release place these inhibitory receptors in a prime position to be feedback modulators of the GH axis. Consistent with our original hypothesis is the fact that an increase in GHRH, which would (in an intact system) lead to an increase in circulating GH, increased sst1 and sst2 mRNA levels, thereby tempering its stimulatory actions on GH release. However, the inhibition of pituitary sst5 expression by GHRH both in vivo and in vitro is inconsistent with our original hypothesis. The opposite action of GHRH on sst5, compared with sst1 and sst2 expression, suggests that the roles of the individual SRIF receptors in the regulation of somatotrope function may vary. Although all SRIF receptors have been shown to be coupled to Gi{alpha}, resulting in suppression of adenylyl cyclase activity, selective activation of the SRIF receptor subtypes have been shown to differentially modulate voltage-sensitive Ca2+ and K+ channels, phospholipase A2 and C, Na+/H+ antiporters, tyrosine and serine/threonine phosphatases, and mitogen-activated protein kinase (5). Another level of complexity that may explain the differential actions of the SRIF receptor subtypes, and thus the need for their differential regulation, is the recent observation that SRIF receptors can form homo- and heterodimers (45) in addition to dimerizing with other G protein-coupled receptors (46). Therefore, the relative balance between SRIF receptor subtypes dimerized with themselves and other G protein-coupled receptors within the same cell may be an important determinant in the activation or suppression of specific effector systems.

Although the results of the present study clearly demonstrate pituitary sst1, sst2, and sst5 expression can be directly mediated by GHRH, the effect of altering the components of the hypothalamic-pituitary-GH axis on pituitary sst4 expression is not as clear cut. Although GH treatment induced a significant rise in sst4 expression in the SDR, there were variable and insignificant differences in sst4 expression between normal and SDRs and after IGF-I treatment. This variability may be related to the level of sst4 expression, which is the lowest of all SRIF receptor subtypes in the rat pituitary, as observed in this study and as shown by others (4, 47). In the human, sst4 is only expressed in the developing pituitary and is not detected in the adult pituitary (12). The low level of expression in the rat pituitary coupled with the observation that sst4-selective agonists fail to suppress GH (42) suggest that sst4 may mediate other aspects of somatotrope function or the function of other pituitary cell types.

In summary, the results of the present study demonstrate that rat pituitary sst1, sst2, sst4, and sst5 mRNA levels are differentially regulated by changes in the hypothalamic-pituitary-GH axis. The sst1 and sst2 mRNA levels are positively correlated, and sst5 mRNA levels are negatively correlated with endogenous GHRH in both the presence and absence of GH, and expression of all three of these SRIF receptor subtypes can be directly modulated by GHRH in vitro. Alterations in these SRIF receptor subtypes after physiological or experimental changes in the GH axis imply that they may be important transducers of negative feedback regulation. However, the differential modulation of these SRIF receptor subtypes suggests that they exert independent and highly selective roles in the modulation of pituitary function.


   Footnotes
 
1 This work was supported by NIH Grant DK-30667 (to R.D.K.) and the Bane Scholar Fund (to L.A.F.). Back

2 Visiting Scientist from the Department of Pharmacology, Kyunghee University School of Medicine, Seoul 130–701, Korea. Back

3 Current address: Department of Medicine, Nippon Medical School, Sendagi 1–1-5, Bunkyo-ku, Tokyo 113, Japan. Back

4 Recipient of the 1998 Endocrine Society Summer Research Fellowship. Back

Received May 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reichlin S 1983 Somatostatin. N Engl J Med 309:1495–1501[Medline]
  2. Reisine T 1995 Somatostatin. Cell Mol Neurobiol 15:597–614[CrossRef][Medline]
  3. Tannenbaum GS, Epelbaum J 1999 Somatostatin. In: Kostyo JL, Goodman HM (eds) Handbook of Physiology: The Endocrine System. Oxford University Press, New York, Oxford, pp 221–265
  4. Epelbaum J, Briard N, Djordjijevic D, Dutour A, Myerhoff W, Oliver C, Slamma A, Viollet C, Zhang J 1998 Characterization of somatostatin receptor subtypes in mammalian pituitary. Ann NY Acad Sci 839:249–253[CrossRef][Medline]
  5. Patel YC 1999 Somatostatin and its receptor family. Front Neuroendocrinol 20:157–198[CrossRef][Medline]
  6. Day R, Dong W, Panetta R, Kracier J, Greenwood MT, Patel YC 1995 Expression of mRNA for somatostatin receptor (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hybridization analysis. Endocrinology 136:5232–5235[Abstract]
  7. O’Carroll AM, Krempels K 1995 Widespread distribution of somatostatin receptor messenger ribonucleic acids in rat pituitary. Endocrinology 136:5224–5227[Abstract]
  8. Kumar U, Laird D, Srikant CB, Escher E, Patel YC 1997 Expression of the five somatostatin receptor (SSTR1–5) subtypes in rat pituitary somatotropes: quantitative analysis by double-label immunoflourescence confocal microscopy. Endocrinology 138:4473–4476[Abstract/Free Full Text]
  9. Mezey E, Hunyady B, Mitra S, Hayes E, Liu Q, Schaeffer J, Schonbrunn A 1998 Cell specific expression of the sst2A and sst5 somatostatin receptors in the rat anterior pituitary. Endocrinology 139:414–419[Abstract/Free Full Text]
  10. Greenman Y, Melmed S 1994 Heterogeneous expression of two somatostatin receptor subtypes in pituitary tumors. J Clin Endocrinol Metab 78:398–403[Abstract]
  11. Greenman Y, Melmed S 1994 Expression of three somatostatin receptor subtypes in pituitary adenomas: evidence for preferential SSTR5 expression in the mammosomatotroph lineage. J Clin Endocrinol Metab 79:724–729[Abstract]
  12. Panetta R, Patel YC 1994 Expression of mRNA for all 5 human somatostatin receptor (hSSTR1–5) in pituitary tumors. Life Sci 56:333–342
  13. Chomczynski P, Downs TR, Frohman LA 1988 Feedback regulation of growth hormone (GH)-releasing hormone gene expression by GH in rat hypothalamus. Mol Endocrinol 2:236–241[Abstract/Free Full Text]
  14. De Gennaro Colonna V, Cattaneo E, Cocchi D, Muller EE, Maggi A 1988 Growth hormone regulation of growth hormone-releasing hormone gene expression. Peptides 9:985–988[CrossRef][Medline]
  15. Bertherat J, Timsit J, Bluet-Pajot MT, Mercadier J-J, Gourdji D, Kordon C, Epelbaum J 1993 Chronic growth hormone (GH) hypersecretion induces reciprocal and reversible changes in mRNA levels from hypothalamic GH-releasing hormone and somatostatin neurons in the rat. J Clin Invest 91:1783–1791
  16. Rogers KV, Vician L, Steiner RA, Clifton DK 1988 The effects of hypophysectomy and growth hormone administration on pre-prosomatostatin messenger ribonucleic acid in the periventricular nucleus of the rat hypothalamus. Endocrinology 122:586–591[Abstract/Free Full Text]
  17. Hurley DL, Phelps CJ 1992 Hypothalamic preprosomatostatin messenger ribonucleic acid expression in mice transgenic for excess or deficient endogenous growth hormone. Endocrinology 130:1809–1815[Abstract/Free Full Text]
  18. Lanzi R, Tannenbaum GS 1992 Time course and mechanism of growth hormone’s negative feedback effect on its own spontaneous release. Endocrinology 130:780–788[Abstract/Free Full Text]
  19. Chan YY, Steiner RA, Clifton DK 1996 Regulation of hypothalamic neuropeptide-Y neurons by growth hormone in the rat. Endocrinology 137:1319–1325[Abstract]
  20. Kamegai J, Unterman TG, Frohman LA, Kineman RD 1998 Hypothalamic/pituitary-axis of the spontaneous dwarf rat: autofeedback regulation of growth hormone (GH) includes suppression of GH releasing-hormone receptor messenger ribonucleic acid. Endocrinology 139:3554–3560[Abstract/Free Full Text]
  21. Kamegai J, Wakabayashi I, Miyamoto K, Unterman TG, Kineman RD, Frohman LA 1998 Growth hormone (GH)-dependent regulation of pituitary GH secretagogue receptor (GHS-R) mRNA levels in the spontaneous dwarf rat. Neuroendocrinology 68:312–318[CrossRef][Medline]
  22. Bruno JF, Xu Y, Song J, Berelowitz M 1994 Pituitary and hypothalamic somatostatin receptor subtype messenger ribonucleic acid expression in the food-deprived and diabetic rat. Endocrinology 135:1787–1792[Abstract]
  23. Takeuchi T, Suzuki H, Sakurai S, Nogami H, Okuma S, Ishikawa H 1990 Molecular mechanism of growth hormone (GH) deficiency in the spontaneous dwarf rat: detection of abnormal splicing of GH messenger ribonucleic acid by the polymerase chain reaction. Endocrinology 126:31–38[Abstract/Free Full Text]
  24. Kineman RD, Kamegai J, Frohman LA 1999 Growth hormone-releasing hormone (GHRH) and the growth hormone secretagogue (GHS), L692,585, differentially modulate rat pituitary GHS receptor (GHS-R) and GHRH receptor (GHRH-R) mRNA levels. Endocrinology 140:3581–3586[Abstract/Free Full Text]
  25. Tannenbaum GS 1981 Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin. Endocrinology 108:76–82[Abstract/Free Full Text]
  26. Joanny P, Peyre G, Steinberg J, Guillaume V, Pesce G, Becquet D, Oliver C 1992 Effect of diabetes on in vivo and in vitro hypothalamic somatostatin release. Neuroendocrinology 55:485–491[Medline]
  27. Tannenbaum GS, Rorstad O, Brazeau P 1979 Effects of prolonged food deprivation on the ultradian growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat. Endocrinology 104:1733–1738[Abstract/Free Full Text]
  28. Vuagnat BA, Pierroz DD, Lalaoui M, Englaro P, Pralong FP, Blum WF, Aubert ML 1998 Evidence for a leptin-neuropeptide Y axis for the regulation of growth hormone secretion in the rat. Neuroendocrinology 67:291–300[CrossRef][Medline]
  29. Cocchi D, De Gennaro Colonna V, Bagnasco M, Bonacci D, Muller EE 1999 Leptin regulates GH secretion in the rat by acting on GHRH and somatostatinergic functions. J Endocrinol 162:95–99[Abstract]
  30. Ishikawa M, Mizobuchi M, Takahashi H, Bando H, Saito S 1997 Somatostatin release as measured by in vivo microdialysis: circadian variation and effect of prolonged food deprivation. Brain Res 749:226–231[CrossRef][Medline]
  31. Bruno JF, Olchovsky D, White JD, Leidy JW, Song J, Berelowitz M 1990 Influence of food deprivation in the rat on hypothalamic expression of growth hormone-releasing factor and somatostatin. Endocrinology 127:2111–2116[Abstract/Free Full Text]
  32. Olchovsky D, Bruno JF, Wood TL, Gelato MC, Leidy Jr JW, Gilbert Jr JM, Berelowitz M 1990 Altered pituitary growth hormone (GH) regulation in streptozotocin-diabetic rats: a combined defect of hypothalamic somatostatin and GH-releasing factor. Endocrinology 126:53–61[Abstract/Free Full Text]
  33. White JD, Olchovsky D, Kershaw M, Berelowitz M 1990 Increased hypothalamic content of preproneuropeptide-Y messenger ribonucleic acid in streptozotocin-diabetic rats. Endocrinology 126:765–772[Abstract/Free Full Text]
  34. Marks JL, Waite K, Li M 1993 Effects of streptozotocin-induced diabetes mellitus and insulin treatment on neuropeptide Y mRNA in the rat hypothalamus. Diabetologica 36:497–502[Medline]
  35. De Gennaro Colonna V, Fidone F, Cocchi D, Muller EE 1993 Feedback effects of growth hormone on growth hormone-releasing hormone and somatostatin are not evident in aged rats. Neurobiol Aging 14:503–507[CrossRef][Medline]
  36. Minami S, Kamegai J, Hasegawa O, Sugihara H, Okada K, Wakabayashi I 1993 Expression of growth hormone receptor gene in rat hypothalamus. J Neuroendocrinol 5:691–696[CrossRef][Medline]
  37. Bruno JF, Xu Y, Berelowitz M 1994 Somatostatin regulates somatostatin receptor subtype mRNA expression in GH3 cells. Biochem Biophys Res Commun 202:1738–1743[CrossRef][Medline]
  38. Lopes MB, Gaylinn BD, Thorner MO, Stoler NH 1997 Growth hormone-releasing hormone receptor mRNA in acromegalic pituitary tumors. Am J Pathol 150:1885–1891[Abstract]
  39. Kraus J, Woltje M, Schonwetter N, Holtt V 1998 Alternative promoter usage and tissue specific expression of the mouse somatostatin receptor 2 gene. FEBS Lett 428:165–170[CrossRef][Medline]
  40. Woltje M, Kraus J, Holtt V 1998 Identification of a functional 3',5'-cyclic adenosine monophosphate response element within the second promoter of the mouse somatostatin receptor type 2 gene. FEBS Lett 440:107–110[CrossRef][Medline]
  41. Patel YC, Greenwood M, Kent G, Panetta R, Srikant CB 1993 Multiple gene transcript of the somatostatin receptor SSTR2: tissue selective distribution and cAMP regulation. Biochem Biophys Res Commun 192:288–294[CrossRef][Medline]
  42. Rohrer SP, Hayes E, Berk SC, Hutchins SM, Shen D-M, Xiong Y, Parma R, Foor P, Mitra SW, Degrado SJ, Shu M, Klopp JM, Cai SJ, Blake A, Chan WW, Pasternak A, Yang L, Patchett A, Smith RG, Chapman KT, Schaeffer JM 1998 Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry. Science 282:737–740[Abstract/Free Full Text]
  43. Kreienkamp H-J, Akgün E, Baumeister H, Meyerhof W, Richter D 1999 Somatostatin receptor subtype 1 modulates basal inhibition of growth hormone release in somatotrophs. FEBS Lett 462:464–466[CrossRef][Medline]
  44. Shimon I, Taylor JE, Dong JZ, Bitonte RA, Kim S, Margan B, Coy DH, Culler MD, Melmed S 1997 Somatostatin receptor subtype specificity in human fetal pituitary cultures. J Clin Invest 99:789–798[Medline]
  45. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC 2000 Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers. J Biol Chem 275:7862–7869[Abstract/Free Full Text]
  46. Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Patel YC 2000 Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 288:154–157[Abstract/Free Full Text]
  47. Kimura N, Tomizawa S, Arai KN, Kimura N 1998 Chronic treatment with estrogen up-regulates expression of sst2 messenger ribonucleic acid (mRNA) but down-regulates expression of sst5 mRNA in rat pituitaries. Endocrinology 139:1573–1580[Abstract/Free Full Text]



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