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Endocrinology Vol. 141, No. 2 498-504
Copyright © 2000 by The Endocrine Society

ARTICLES

Selective Impairment of Corticotropin-Releasing Factor1 (CRF1) Receptor-Mediated Function Using CRF Coupled to Saporin1

Dominique Maciejewski-Lenoir2, Steve C. Heinrichs2, Xin-Jun Liu, Nicholas Ling, Anh Tucker, Qiu Xie, Douglas A. Lappi and Dimitri E. Grigoriadis

Neurocrine Biosciences, Inc. (D.M.-L.), San Diego, California 92121; Department of Psychology, Boston College (S.C.H.), Chestnut Hill, Massachusetts 02467; and Advanced Targeting Systems (D.A.L.), San Diego, California 92121

Address all correspondence and requests for reprints to: D. Maciejewski-Lenoir, Ph.D., Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, California 92121. E-mail: dmaciejewski{at}neurocrine.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1–100 nM) produced concentration-dependent and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on CRF2{alpha} receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12–41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors, CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1–10 nM) stimulated LH release was also assessed in the same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRF, a 41-residue neuropeptide, mediates the body’s endocrine, behavioral, autonomic, and immune responses to stress (1). The action of CRF is mediated by its receptors, which have been extensively characterized. Two G protein-coupled receptor subtypes, CRF1 (2, 3, 4) and CRF2{alpha}/CRF2ß (5), were recently cloned. These two receptors show different affinities for various CRF family peptide ligands (1). In particular, native rat/human (r/h) CRF has a higher affinity for the CRF1 than for the CRF2 receptor. This differential affinity has been argued to provide some measure of functional receptor selectivity when interpreting the effects of CRF agonist administration (6).

To directly examine the consequences of long term CRF system impairment, a novel protein conjugate (CRF-SAP) was designed by joining native r/hCRF to a ribosome-inactivating protein, saporin (SAP) (7). This approach stems from several studies in the literature reporting impairment of CRF system function using both unconjugated (8) and conjugated (9) toxins. In an earlier report, Schwartz et al. (9) reported on the use of CRF conjugated to gelonin (20 mol [Nle21,28,Arg36]rCRF/mol gelonin.), a ribosome-inactivating protein from Gelonium multiflorum. We have selected saporin, used extensively in vivo as a targeted toxin for research use (10, 11, 12, 13), instead of gelonin for several reasons. First, saporin is 10-fold more active than gelonin in the inhibition of cell-free protein synthesis (14). Second, unlike gelonin, SAP is not glycosylated. Glycosylation of a ribosome-inactivating protein has been reported to have deleterious effects in animals due to greatly reduced pharmacokinetics for in vivo applications (15, 16). Thus, the present CRF-SAP conjugate would be expected to target only CRF receptor-expressing cells and deliver the saporin toxin inside those cells through a receptor internalization mechanism. The activity of saporin, namely the inhibition of ribosome activity and de novo protein synthesis, can only be achieved after internalization (14). CRF has been shown to be internalized upon ligand binding (17). Although the internalization of the CRF-SAP construct has not been physically observed, it has been clearly demonstrated for other peptide receptor systems. For example, the internalization of substance P attached to saporin has been demonstrated upon binding to its G protein-coupled receptor (18). This process leads to cell death and loss of neurotransmitter function (11, 12).

To ensure that CRF-SAP possesses the desirable biological properties, we have characterized the receptor binding profile of the synthetic conjugate using CRF1-transfected L cells and CRF2{alpha}-transfected Chinese hamster ovary (CHO) cells. In addition, the cytotoxicity of CRF-SAP was assessed in vitro in both CRF1-transfected L cells and CRF2{alpha}-transfected CHO cells. We also used primary cultures of rat anterior pituitary cells to demonstrate the selective toxicity of CRF-SAP on corticotrophs by measuring CRF-induced ACTH release and GnRH-induced LH release. Results from these measures were used to evaluate the potential utility of CRF-SAP as a potent, selective, and effective tool for long term in vivo impairment of CRF1 receptor-expressing cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs and peptides
r/hCRF and [D-Phe12]r/hCRF-(12–41)NH2 (D-Phe-CRF) were synthesized in-house using solid phase methodology on a peptide synthesizer (model 990, Beckman Coulter, Inc., Palo Alto, CA). Saporin and r/hCRF-(1–41)NH2/saporin (CRF-SAP) were obtained from Advanced Targeting Systems (San Diego, CA). CRF-SAP was synthesized by conjugation of a CRF analog that contains the full r/hCRF-amide sequence. A disulfide bond connects the two molecules. There is 1 mol CRF/mol saporin in the construct. [125I]Tyr0-sauvagine was purchased from NEN Life Science Products-DuPont (Boston, MA). NBI 27914 was also synthesized at Neurocrine Biosciences (San Diego, CA). Phorbol myristic acid was purchased from Sigma (St. Louis, MO).

CRF1 CRF2{alpha} and CRF-binding protein (CRF-BP) binding assays
CRF receptor binding affinities were determined from competition curves for saporin, CRF, and CRF-SAP using [125I]Tyr0-sauvagine as described previously (19). Briefly, cells expressing the respective receptors were thawed on ice and diluted in 5–7 ml ice-cold PBS containing 10 mM MgCl2 and 2 mM EGTA, pH 7.0, at 22 C (tissue buffer) and homogenized at 25,000 rpm for 15 sec on ice using a Polytron (Brinkmann Instruments, Inc., Westbury, NY) tissue homogenizer. Membrane homogenates were then washed twice by centrifugation (30,000 x g for 10 min each time at 4 C), and the final pellets were resuspended in buffer to a working concentration of approximately 1.2 mg/ml protein. Typically, 2.5 x 106 CHO cells yielded 1 mg protein, with the final protein concentration in the assay being 50 µg/tube. For competition studies, membranes were incubated for 2 h with radiolabeled sauvagine (200 pM) and 1-pM to 1-µM concentrations of competing peptides. The reaction was terminated by centrifugation, and the resulting pellets were monitored for radioactivity. Inhibitory coefficients were determined for saporin, CRF, and CRF-SAP at the CRF-BP using an enzyme-linked immunosorbant assay as described previously (20).

In vitro cell survival assay
Confluent transfected cells were grown for 24–96 h in the presence of various concentrations of CRF-SAP or other compounds. Cell death was determined with an assay kit (Roche Molecular Biochemicals, Indianapolis, IN) by measuring the release of lactate dehydrogenase (LDH) in the cell supernatants. The cells were further incubated for an additional 4 h in the presence of tetrazolium salt [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)] to assess mitochondrial metabolic function through the formation of MTT formazan (absorbance was measured at 590 nm; Roche Molecular Biochemicals).

Pituitary ACTH and LH release assays
Pituitaries were removed from female adult rats (50 days old); digested by collagenase, dispase, and neuraminidase; and gently triturated to yield a single cell suspension. The cells were plated in BBM-T [McCoy’s 5A from Life Technologies, Inc. (Gaithersburg, MD), containing 2.4 g/liter HEPES, 2 g/liter BSA, 10 mg/liter transferrin, 50,000 IU/liter penicillin and streptomycin, 1 µg/liter insulin, 0.1 µg/liter epidermal growth factor, 0.4 µg/liter T3, 0.7 µg/liter PTH, and 10 µg/liter glucagon] medium supplemented with 3% FBS at a density of 1–2 x 104 cells/cm2 and cultured for 3 days at 37 C in an atmosphere of 7.5% CO2. To begin the assay, the cells were then incubated for 24–96 h with CRF-SAP or other compounds. After washing the cells with serum-free medium, they were then incubated for 4 h with 1 nM CRF in serum-free medium to stimulate ACTH release or for 4 h with 1–10 nM GnRH to stimulate LH release. After incubation, the medium was harvested, and the respective amounts of pituitary hormones were determined by RIA.

ACTH RIA
Briefly, the conditioned medium was diluted 3-fold with RIA buffer (19 mM monobasic and 81 mM dibasic sodium phosphate, 0.05 M NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.01% NaN3). One hundred microliters of rabbit anti-rACTH serum (diluted 1:3,000) and 100 µl RIA buffer were added to 100-µl samples or rACTH standards. The mixture was incubated overnight at 4 C. One hundred microliters of competing 125I-labeled rACTH (~20,000 cpm) were then added to the reaction, and the mixture was further incubated overnight at 4 C. Finally, 100 µl goat antirabbit IgG antibody (1:20) and 100 µl normal rabbit serum (1:100) were added, and the reaction mix was incubated for an additional 24 h at 4 C. The tubes were centrifuged at 3,000 rpm for 30 min, the supernatants were removed, and the radioactivity was counted in a {gamma}-counter.

LH RIA
Briefly, the conditioned medium was diluted 5-fold with RIA buffer (10 mM sodium phosphate buffer, 0.15 M NaCl, 1% BSA, and 0.01% NaN3, pH 7.5). One hundred microliters of [125I]rLH (30,000 cpm), 100 µl rabbit anti-rLH antibody (diluted 1:187,500; from National Hormone and Pituitary Program), and 100 µl RIA buffer were added to 100-µl samples or rLH standards. The mixture was incubated overnight at room temperature. One hundred microliters of goat antirabbit IgG antibody (1:20) and 100 µl normal rabbit serum (1:100) were then added, and the reaction mix was incubated for an additional 3 h at room temperature. The tubes were finally centrifuged at 3000 rpm for 30 min, the supernatants were removed, and the radioactivity in the resulting pellets was monitored in a {gamma}-counter.

Statistical analysis
Receptor competition assays were repeated twice, and radioligand binding analyses were performed using Prism (GraphPad Software, Inc., San Diego, CA). Each curve was fit using a nonlinear least squares curve fitting algorithm followed by the Cheng-Prusoff correction for the conversion of EC50 values to Ki based on the concentration of radioligand used in each assay (21). Triplicate determinations of cytotoxicity were averaged, and each experiment was repeated. ACTH release measurements were performed in quadruplicate. The results are described as the mean ± SEM of one experiment repeated four times. Statistical analysis was performed using two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Competition binding assays
Both CRF receptor agonists CRF and CRF-SAP bound the two CRF receptor subtypes and the CRF-BP with similar affinity and exhibited the typical rank order affinity profile CRF-BP > CRF1 > CRF2{alpha} (see Table 1Go). In contrast, although CRF-SAP demonstrated relatively high affinities at the CRF1 and CRF2{alpha} receptors as did the unconjugated CRF, the saporin protein alone did not appear to bind to any of the receptors at concentrations up to 10 µM (see Fig. 1Go). Table 1Go compares the relative affinities of the CRF family of ligands employed in the present studies with those of other peptide standards at the CRF receptors and binding protein.


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  		Table 1. Ligand affinities at CRF1 and CRF2{alpha} receptors and CRF-binding protein

 


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  		Figure 1. Competition of various peptides with [125I]Tyr0-sauvagine binding in CRF receptor-expressing cell lines. Human CRF1-transfected L cell (top) or CRF2{alpha}-transfected CHO cell (bottom) membranes were incubated with [125I]Tyr0-sauvagine (225 pM) in the presence of various concentrations of peptides. Peptides, unconjugated SAP, CRF, or CRF-SAP were examined for their ability to inhibit specific binding. Each point represents duplicate determinations, and the data were repeated with similar results. Data were expressed as the percent specifically bound and typically represented a total binding of [125I]sauvagine of 8000 cpm and nonspecific counts of 1000–1200 cpm (competed with 1 µM r/hCRF). This yielded typical specific binding of more than 85% in all experiments. All data were analyzed using the curve-fitting algorithms in Prism as described in detail in Materials and Methods (GraphPad Software, Inc.).

 
Cytotoxicity assay
The first in vitro validation of the cytotoxicity of CRF-SAP was performed using CRF1-transfected LtK- cells and CRF2{alpha}-transfected CHO cells. Incubation of both cell types with increasing doses of CRF-SAP over 1, 2, or 3 days resulted in a concentration-dependent and progressive cell death over time. In CRF1-transfected L cells there was a dose- and time-dependent increase in LDH levels, with maximal toxicity obtained after 3 days with 100 nM CRF-SAP (see Fig. 2aGo). LDH release into the medium was over 3-fold higher than that in control samples, whereas MTT formazan metabolization was reduced by more than 50% (Fig. 2bGo), indicating both an increase in cell death and a decrease in cell viability.



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  		Figure 2. CRF-SAP cytotoxicity in L cells transfected with CRF1 receptors (A and B) or CHO cells transfected with CRF2{alpha} receptors (C and D). Confluent transfected cells were grown for 1–3 days in the presence of varying concentrations of CRF-SAP. Cell death was assessed in the supernatant by measuring the release of LDH (A and C). The cells were then incubated for an additional 4 h in the presence of tetrazolium salt (MTT) to assess mitochondrial metabolic function (B and D).

 
On the other hand, CRF2{alpha}-transfected CHO cells did not show any significant increase in LDH release at any time when tested at doses up to 100 nM (Fig. 2cGo). In fact, we observed a paradoxical increase in MTT values at 1 and 10 nM CRF-SAP that did not occur at 100 nM (Fig. 2dGo). In untransfected LtK- or CHO cells, treatment with CRF-SAP (100 nM) did not exhibit any significant cytotoxicity (data not shown). These results strongly indicate that at these concentrations, CRF-SAP targets only cells expressing the CRF1 receptor. To demonstrate the pharmacological significance of the cytotoxicity, cell death was assessed after 3-day treatment with CRF-SAP in conjunction with selective or nonselective CRF antagonists. As demonstrated in Fig. 3aGo, the toxic effect of CRF-SAP on CRF1-transfected L cells was inhibited by both the nonselective peptide CRF receptor antagonist, D-Phe-CRF and the selective CRF1 receptor nonpeptide antagonist NBI 27914. Figure 3bGo demonstrates that in CRF2 receptor-transfected cells, CRF-SAP also caused cytotoxicity that could be protected by D-Phe-CRF, however at much higher concentrations (25- to 50-fold), confirming the lower affinity of r/hCRF for the CRF2 receptor subtype (see Fig. 3bGo). These data suggest that there is a preferential toxicity of CRF-SAP on CRF1 receptor-expressing cells.



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  		Figure 3. Reversal of the cytotoxic effect of CRF-SAP in CRF1 (a)- or CRF2{alpha} (b)-expressing cells by CRF receptor antagonists. Confluent CRF1-transfected L cells and CRF2a-transfected CHO cells were grown for 3 days in the presence of varying concentrations of CRF-SAP and a nonselective CRF receptor antagonist (D-Phe-CRF) or a CRF1-selective nonpeptide receptor antagonist (NBI 27914). Cell death was assessed in the supernatant by measuring the release of LDH.

 
ACTH release assay
The biological consequences of treating CRF receptor-expressing cells with CRF-SAP were further investigated in a primary pituitary cell culture system. In this system, a small portion of the cells (2–5%) express CRF receptors (corticotropes) and these cells respond to CRF stimulation by releasing ACTH. Incubation of the cells for 1, 2, or 3 days with increasing concentrations of CRF-SAP resulted in a dose-dependent inhibition of the ability of CRF to stimulate ACTH release (Fig. 4Go). At higher doses (> 10 nM), CRF-SAP completely abolished CRF-induced ACTH release, and at 100 nM, the reduction was below the basal release level. The cytotoxic effect of CRF-SAP could be inhibited by coincubating the cells with the CRF receptor peptide antagonist, D-Phe-CRF. In this treatment, cells were exposed to 10 nM CRF-SAP for 3 days in either the presence or absence of D-Phe-CRF, and after washing, the cells were allowed to recover for an additional 2 days. After the recovery period, the cells were stimulated with 1 nM CRF and assessed for their ability to release ACTH. As shown in Fig. 5Go, treatment with CRF-SAP alone abolished the CRF stimulatory effect, and this toxicity could be dose dependently blocked with the concomitant administration of D-Phe-CRF. D-Phe-CRF alone had no effect on the ability of CRF to elicit release of ACTH from the cells after recovery. To discriminate between CRF-mediated receptor desensitization and CRF-SAP-mediated cell toxicity, pituitary cells were incubated for 3 days with 10 nM CRF-SAP, CRF, or SAP alone and then stimulated with either CRF or phorbol myristic acid (PMA). PMA has been shown to stimulate the release of ACTH in a nonreceptor-mediated fashion, through direct activation of protein kinase C (22). Figure 6Go shows that indeed, preincubation of the cells with CRF alone blunted the subsequent CRF-elicited ACTH release, an effect presumably caused by either receptor occupancy or desensitization of the CRF1 receptor. However, cells pretreated with CRF could still elicit a normal ACTH response to PMA, indicating that the cells remained viable. As expected, both CRF- and PMA-induced ACTH release was totally abolished by pretreatment of the cells with CRF-SAP. Importantly, SAP alone only mildly inhibited ACTH release, confirming that the toxicity is CRF1 receptor mediated.



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  		Figure 4. Attenuation of CRF-induced ACTH release by CRF-SAP in primary pituitary cells. Pituitary cells removed from adult rats were enzymatically digested and plated for 3 days before incubation with varying concentrations of CRF-SAP for an additional 1–3 days. The cells were then stimulated with 1 nM CRF for 4 h, and ACTH release was assessed by RIA.

 


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  		Figure 5. Antagonism of CRF-SAP cytotoxicity by the CRF receptor antagonist D-Phe-CRF. Pituitary primary cell cultures were grown for 3 days and incubated for an additional 3 days in the presence or absence of CRF-SAP and varying concentrations of the nonselective CRF receptor antagonist, D-Phe-CRF. The cells were washed and allowed to recover for an additional 2 days. Cells were then stimulated with CRF (1 nM), and ACTH release was measured by RIA.

 


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  		Figure 6. CRF-SAP-induced cytotoxicity abolishes CRF- or PMA-elicited ACTH release. Pituitary primary cell cultures were grown for 3 days, then incubated for 3 days with CRF-SAP, CRF, or SAP alone. The cultures were stimulated with CRF (1 nM) or PMA (100 nM), and ACTH release was assessed by RIA.

 
To further demonstrate the CRF-SAP specificity, we tested the ability of GnRH to stimulate LH release in the same pituitary cell cultures. Figure 7Go clearly demonstrates that although corticotropes were susceptible to the toxic actions of the CRF-SAP conjugate, gonadotropes in the same cultures were not affected. The slight decrease in the ability of GnRH to stimulate LH release from treated cultures compared with control was equivalent for the conjugated and unconjugated forms of SAP, suggesting that SAP alone may cause some nonspecific toxicity.



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  		Figure 7. CRF-SAP cytotoxicity does not affect GnRH-induced LH release. Pituitary primary cell cultures were grown for 3 days, then incubated for 3 days with CRF-SAP, CRF, or SAP alone. The cultures were then stimulated with GnRH (10 nM) and assessed for their ability to release LH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this in vitro study we have demonstrated that a CRF-toxin conjugate protein binds CRF1 receptors with high affinity, affects cell viability in CRF1-transfected L cells, and impairs function in pituitary cells expressing CRF1 receptors. Interestingly, both CRF and CRF-SAP bound CRF-binding sites with the same rank order of affinity: CRF-BP > CRF1 > CRF2{alpha}. Moreover, both CRF and CRF-SAP exhibited low nanomolar affinities for CRF1 and 30-fold or more decreased affinities for CRF2{alpha}. Thus, the preferential interaction of CRF-SAP with CRF1 vs. CRF2 receptors in the present studies appears to follow the pharmacological rank order profile of r/hCRF for the two receptor subtypes and appears not to be altered by its linkage to saporin. Furthermore, toxin targeting to other CRF receptors may be accomplished by conjugation of saporin to other CRF family ligands that possess higher affinity for their respective receptors (refer to Table 1Go). Exposure of cultured primary pituitary cells to CRF-SAP resulted in inhibition of ACTH release. This is probably caused by toxicity of CRF-SAP to the corticotropes. The fact that GnRH could still elicit a response by stimulating LH release from these same cultures indicates the specificity of the toxic response. The absence of cytotoxicity in CRF-SAP-treated wild-type L-cells coupled with the lower sensitivity in CRF2{alpha} receptor-transfected CHO cells, further demonstrates that the impairment in cellular function caused by 10–100 nM CRF-SAP is CRF1 receptor mediated. In addition, both the cytotoxicity and suppression of CRF-stimulated ACTH release were competitively antagonized by coincubation with either the peptide CRF receptor antagonist D-Phe-CRF or the nonpeptide CRF1 receptor-selective antagonist, NBI 27914.

The mechanism of action for CRF-SAP in modifying ACTH release was distinct from that of the two separate peptides used to form the conjugate. CRF alone was able to desensitize the CRF1 receptor, whereas SAP induces some mild, nonselective cytotoxicity. In contrast, CRF-SAP bound to CRF receptors with high affinity and impaired corticotroph function. SAP alone exhibited neither affinity for CRF receptors nor impaired basal, CRF- or PMA-induced ACTH release in cultured pituitary cells. Prior exposure of the cells to 10 nM CRF alone blunted the ACTH response to additional CRF, but this effect was most probably caused by CRF receptor desensitization, as direct stimulation with PMA elicited a normal ACTH response in cells preincubated with CRF. In contrast, 3-day preincubation with 10 nM CRF-SAP abolished both CRF- and PMA-induced ACTH release and even diminished basal ACTH secretion. This indicated that the toxin was selectively destroying the corticotropes expressing the CRF1 receptor.

Interestingly, the saporin toxin alone did show some toxicity, as measured by LDH release in both CRF1-transfected cells and pituitary cells. This toxic effect was not blocked by CRF antagonists and was not present in nondividing primary hypothalamic cultures, suggesting an in vitro mitotoxic effect on dividing cells (data not shown). Although we have not observed any dissociation of CRF from saporin (Lappi, D., personal communication), this possibility cannot be excluded. However, previous studies of peptide ligand-toxin conjugates have shown that the products were stable with prolonged efficacy in vivo (18). In addition, saporin conjugates with similar chemistry, but linked to antibodies, have shown efficacy and stability in in vivo studies using nonhuman primate models (23). Prior in vivo experiments showed that saporin alone had moderate toxicity in mice (LD50 = 6 mg/kg), as it was rapidly cleared from the bloodstream by the kidneys (7). In contrast, when it was linked to monoclonal antibodies specific for immune cells (24, 25) or coupled to basic fibroblast growth factor (13), specific cellular toxicity was evident. Thus, our present results are consistent with previous models of immmunotargeting and receptor targeting of toxins. The efficacy of a monoclonal CRF antibody/ricin mixture in impairing the function of the CRF system has been recently documented in studies using measures such as selective intracellular uptake of the antibody/toxin. In these studies it was shown that the reactivity of the hypothalamic-pituitary-adrenal axis to stress and compensatory changes in CRF tone were attenuated after central administration of the immunotoxin (26, 27).

The present studies support the potential utility of the targeted toxin approach as a tool for receptor-selective impairment of the CRF system function. The receptor selectivity, robust efficacy, and cytotoxic nature of CRF-SAP suggest its suitability as a complement to CRF receptor knockout and antisense knockdown in determining the physiological relevance of CRF neurobiology in vivo.


   Acknowledgments
 
We thank Nicola Duggan, Marge Lorang, and On Khongsaly for skillful technical assistance.


   Footnotes
 
1 A portion of this work was presented at the 28th Annual Meeting of the Society for Neuroscience, Los Angeles, California, November 1998. Back

2 D.M.-L. and S.C.H. contributed equally to the present studies. Back

Received May 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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