Propagação in vitro de Hypericum cordatum (Vell. ) N. Robson (Clusiaceae) e análise fitoquímica de seus compostos secundários / In vitro propagation of Hypericum cordatum (Vell. Conc. ) N. Robson (Clusiaceae) and phytochemical analysis of its secondary compounds

Hypericum cordatum, planta com possível atividade medicinal foi analisada no presente estudo quanto a sua propagação in vitro e seus principais compostos secundários em comparação com Hypericum perforatum, espécie medicinal utilizada como antidepressivo. Diante das dificuldades que ocorrem na coleta e sua multiplicação por sementes ou via estacas caulinares, iniciou-se a propagação in vitro tendo como resultados: que a indução e o crescimento de brotações foram estimulados pelo regulador 6-benziladenina (2,0 mg L-1), que houve indução de raízes por ácido indol-butírico (0,5 mg L-1), e que as baixas concentrações de auxinas, ácido 2,4-diclorofenoxiacético, ou ácido naftalenoacético (0,01 a 0,4 mg L-1) induziram a formação de calos sendo uma alternativa viável para a multiplicação desta espécie in vitro. Constatou-se com base nas análises bioquímicas e cromatográficas realizadas nesta fase de desenvolvimento das plantas mantidas em culturas in vitro, que as mesmas não contêm hipericina. No entanto, pode-se atribuir o potencial medicinal de H. cordatum às outras substâncias que também possuem importantes atividades biológicas, tais como a xantona e o ácido clorogênico sintetizados nas glândulas foliares de H. cordatum.

Hypericum cordatum, a plant with possible medicinal activity, was analyzed in this study for its in vitro propagation and main secondary compounds, compared to Hypericum perforatum, a medicinal species used as antidepressant. Considering the difficulties that occur in its collection and multiplication through seeds or stem cuttings, in vitro propagation was started and showed the following results: the induction and the growth of sprouts were stimulated by the plant regulator 6-benzyladenine (2.0 mg L-1), there was root induction by indolebutyric acid (0.5 mg L-1) and the low concentrations of auxins, 2,4-dichlorophenoxyacetic acid or naphthaleneacetic acid (0.01 to 0.4 mg L-1) induced callus formation, constituting a viable alternative for the in vitro multiplication of this species. Biochemical and chromatographic analyses carried out at this developmental stage of plants kept in in vitro cultures indicated that they do not contain hypericin. However, the medicinal role of H. cordatum can be attributed to other substances that also have important biological activities, such as xanthone and chlorogenic acid, both synthesized in the leaf glands of H. cordatum.

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Human astrocytoma U138MG cells express predominantly type-A endothelin receptor.

Endothelin-1 (ET-1) binding to human astrocytoma U138MG cells was time-dependent, and bound [125I]ET-1 was difficult to dissociate. The B(max) and Kd values of [125I]ET-1 binding were 70 fmol/mg and 0.07 nM, respectively. Interestingly, different from other astrocytoma cells and astrocytes, the U138MG cells expressed predominantly ETA receptor as shown by RT-PCR results and binding studies. ET-1, FR139317, BQ123, PD142893 and Ro46-2005 inhibited specific [125I]ET-1 binding with Ki values of 0.10, 0.53, 4.3, 22, and 320 nM, respectively. ETB selective ligands ET-3 and IRL1620 were much less potent. The inhibitory effects of antagonists BQ123 and PD142893 on [125I]ET-1 binding diminished following the incubation time. ET-1 binding caused a modest stimulation in phosphatidylinositol hydrolysis with an EC50 value of 24 nM. In comparison to the human U373MG cells, ET-1-induced receptor internalization in U138MG cells was less efficient with 42% of bound ET-1 internalized after 30 min of incubation. These results imply that human astrocytoma cells/astrocytes are able to express either ETA or ETB receptor under different pathophysiological conditions.

Persistent activation of the dopamine D1 receptor contributes to prolonged receptor desensitization: studies with A-77636.

A-77636 is a dopamine (DA) D1 receptor-selective agonist that was previously shown to elicit beneficial responses in animal models of Parkinson's disease (PD) (Kebabian et al.: Eur. J. Pharmacol. 229: 203, 1992). However, A-77636 is of limited potential for PD therapy because it induces rapid tolerance in vivo. To understand the basis of rapid onset of tolerance to the compound, we conducted studies to compare the in vitro properties of A-77636 and A-81686; the latter is a structurally related D1 agonist that did not induce significant tolerance in vivo under similar experimental conditions. With SK-N-MC, a neuroblastoma cell line, as an in vitro model for the D1 receptor, significant differences in D1 receptor function were noted after pretreatment with the two compounds. Specifically, 1-hr pretreatment with A-77636 resulted in significant residual cAMP production, even after the drug solution was removed and the cells were washed. The residual cAMP activity was selectively inhibited by SCH 23390, a selective D1 antagonist. The residual cAMP activity declined with pretreatment time, and after 4-hr pretreatment, little residual cAMP production was observed. Cotreatment of SK-N-MC cells with SCH 23390 and A-77636 did not prevent residual cAMP production by A-77636. In contrast, A-81686 did not elicit residual cAMP production is SK-N-MC cells. Although A-77636 treated cells were devoid of agonist response 4 hr after drug removal, A-81686-treated cells exhibited significant cAMP response after drug removal. Preincubation of rat striatal membranes with A-77636 resulted in a large decrease in D1 receptor binding, despite repeated washings, whereas A-81686 pretreatment caused only a small reduction in D1 receptor binding. On the basis of the present data, we conclude that A-77636 dissociates slowly from the D1 receptor. The continued activation of the D1 receptor by A-77636 leads to inability of the receptor to recover its responsivity, which may explain its long duration of action and its ability to induce rapid behavioral tolerance in vivo.

Characterization of cloned human dopamine D1 receptor-mediated calcium release in 293 cells.

Dopamine (DA) D1 receptors are generally known to couple only to Gs and cAMP production. Recently, D1 receptors expressed in mouse Ltk- cells have been shown to induce cAMP production, phosphoinositide (PI) hydrolysis, and calcium mobilization [Mol. Endocrinol. 6: 1815-1824 (1992)]. To further evaluate second messenger systems that could be activated by the D1 receptor, we examined the effects of DA, (R)-(+)-SKF-38393, and DA antagonists on cAMP production and calcium release in human embryonic kidney 293 cells stably expressing three different levels (Bmax = 0.12, 1.4, and 23 pmol/mg of protein) of the human D1 receptor. DA and (R)-(+)-SKF-38393 activated cAMP production and calcium release in all three D1-293 clones, and their potency was proportional to receptor density. The efficacy of SKF-38393 was also increased with receptor density in both cAMP and calcium studies. The effect of DA on calcium release consisted of a transient peak response (< 20 sec) that declined to an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive plateau level above the base-line (>5 min). The effect of DA on cAMP and calcium release was selectively inhibited by SCH23390, a selective D1 antagonist, and not by spiperone, a selective D2 antagonist. DA did not induce PI hydrolysis in any of the three receptor-expressing clones. A 24-hr pretreatment with cholera toxin (2 micrograms/ml) greatly attenuated the effect of DA on cAMP formation and calcium release. To address how DA could activate calcium release without enhancing PI hydrolysis, the effects of forskolin, thapsigargin, and isoproterenol (Iso) were studied. Similarly to the effects of DA, forskolin and Iso stimulated cAMP production and calcium release from D1-293 cells. Cells that were stimulated with Iso or forskolin showed a reduced response to subsequent addition of DA. Pretreatment of D1-293 cells with thapsigargin, a selective Ca2+-ATPase inhibitor, elicited calcium release from the inositol-1, 4, 5-trisphosphate-sensitive calcium store and attenuated the response to subsequent addition of DA. Carbachol stimulated PI hydrolysis and calcium release but had little effect on cAMP production. Prestimulation with carbachol abolished the calcium response to DA, Iso, or forskolin. These studies indicate that D1 receptor-mediated calcium mobilization in 293 cells is dependent on cAMP production and the cAMP-dependent calcium store is part of the inositol-1,4,5-trisphosphate-sensitive calcium pool.

Novel Asp32-replacement tetrapeptide analogues as potent and selective CCK-A agonists.

A series of novel CCK tetrapeptide analogues of the general formula Boc-Trp-Lys(Tac)-N(R)-(CH2)nCON(R')Phe-NH2 (Tac = o-tolylaminocarbonyl), where R,R' = H or Me and n = 1-5, have been synthesized and tested. These analogues, which lack an acidic residue at the penultimate position, demonstrated surprisingly high CCK-A receptor affinity and selectivity. The effect of N-methylation pattern on CCK-A receptor affinity showed consistent trends for analogues in which n = 1, 2, or 3, with the di-N-methylated analogues having the highest affinity in each case. However, none of these analogues had full agonist activity, as measured by percent maximal PI hydrolysis. Two conformationally constrained analogues also demonstrated high CCK-A receptor affinity and selectivity, as well as nearly maximal agonist activity. In addition, one of these conformationally-constrained analogues demonstrated anorectic activity in rats.

CCK-A-selective tetrapeptides containing lys(N epsilon)-amide residues: favorable in vivo and in vitro effects of N-methylation at the aspartyl residue.

Previous structure-activity studies on a series of CCK-A selective tetrapeptide agonists, typified by A-71623 (Boc-Trp-Lys(CONH-Ph-o-Me)-Asp-(N-Me)Phe-NH2), have shown that replacement of the Lys(N epsilon-carbamoyl) substituent with N epsilon-acyl substituents resulted in partial agonists with moderate to high affinities for the CCK-A receptor and that replacement of the C-terminal dipeptide with either (N-Me)Asp-Phe or (N-Me)Asp-(N-Me)Phe was highly favorable to in vitro and in vivo CCK activity. The present study demonstrates that although analogues in the epsilon-amide series that are N-methylated at the Phe position are weakly active or inactive in an in vivo rat appetite suppression assay, incorporation of (N-Me)Asp or (N-Me)Asp-(N-Me)Phe modifications in this series results in analogues with markedly improved in vivo activity. In in vitro assays, there is minimal effect of N-methylation pattern on binding affinity, whereas there is a trend toward improved functional activity in the phosphatidylinositol hydrolysis assay in analogues containing (N-Me)Asp.

Tetrapeptide CCK-A agonists: effect of backbone N-methylations on in vitro and in vivo CCK activity.

N-Methylation of backbone amide bonds was conducted on a tetrapeptide that had been identified previously (Shiosaki, K.; et al. J. Med. Chem. 1991, 34, 2387-2842) as a potent and selective CCK-A agonist. N alpha-Methylation at the position corresponding to Asp32 (CCK-33 numbering) was consistent with high affinity, efficacy, and selectivity for the CCK-A receptor. Combination of this (N-Me)Asp with the (N-Me)Phe modification also provided a highly active analogue. The observation of parallel structure-binding affinity profiles with respect to sites of N-methylation in the C-terminal regions of tetrapeptide vs heptapeptide CCK analogues suggests that the two series interact similarly with the CCK-A receptor.

Novel CCK analogues and bombesin: a detailed analysis between phosphoinositide breakdown and high-dose inhibition of pancreatic enzyme secretion in three rodent species.

Cholecystokinin octapeptide (26-33) (CCK-8) stimulates pancreatic amylase secretion in a biphasic manner. Amylase secretion is stimulated in a dose-dependent manner up to a maximal level, but reduced secretion is observed at supramaximal concentrations. The downward portion of the dose-response curve has been referred to as "high-dose" inhibition. Recent studies with CCK-8 and Boc-Tyr(SO3H)-Nle-Gyl-Trp-Nle-Asp-2-phenylethylester (JMV-180) using rat acini have suggested that activation of the low-affinity CCK receptor leads to enhanced phosphoinositide (PI) breakdown, which in turn is responsible for high-dose inhibition of enzyme release. However, the secretory effect of JMV-180 varied considerably between rat and mouse. To explore further the relationship between PI breakdown and high-dose inhibition, we compared the effects of JMV-180 as well as the novel cholecystokinin tetrapeptide (30-33) Trp-Met-Asp-Phe-NH2 analogs A-70874 and A-57282, using rat, mouse and guinea pig pancreatic acini. The maximal secretory activity of CCK-8 was lowest (approximately 10% of total cellular amylase) in mouse, as compared with guinea pig and rat (approximately 15-20% of total amylase). The efficacies of A-70874, A-57282 and JMV-180 for stimulation of PI breakdown, relative to CCK-8, were 100, 100 and 45%, respectively, in mouse; 95, 70 and 20%, respectively, in rat and 75, 40 and 0%, respectively, in guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetrapeptide CCK agonists: structure-activity studies on modifications at the N-terminus.

We had reported earlier on a novel series of potent and selective tetrapeptide cholecystokinin-A (CCK-A) agonists of the general structure Boc-Trp-Lys[epsilon-Y]-Asp-N(R)PheNH2 [Y = amides, ureas; R = H, Me] that were potent anorectic agents in rats. In an effort to optimize the potency, selectivity, stability, and efficacy of our lead candidate A-71623 [R = Me, Y = o-tolylaminocarbonyl; Tac] toward development of a clinical candidate, we have explored a series of analogues in which the N-terminal Boc functionality was systematically replaced with various amides, ureas, carbamates, and sulfonamides of differing size, hydrophobicity, and stereoelectronic properties. In general, these analogues maintained good potency and selectivity for the CCK-A receptor (guinea pig pancreas), as well as potent anorectic activity in rats. Those analogues exhibiting equal or superior activity compared to A-71623 but differing physicochemical properties may represent superior drug candidates.

Peripheral cholecystokinin type A receptors mediate oxytocin secretion in vivo.

Cholecystokinin-octapeptide (CCK8) administered intraperitoneally (i.p.) in rats induces a rapid elevation in serum oxytocin (OT). The receptor subtype mediating this action of CCK was investigated with selective CCK-A and CCK-B receptor agonists and antagonists. CCK8 and A-71623, a potent CCK-A selective agonist, were similar in efficacy and potency for stimulating OT secretion. Both compounds at 10 nmol/kg elicited approximately one-half the response of 100 nmol/kg, which elevated serum OT to approx. 20 to 30-fold above basal level. The potent CCK-B selective agonist, A-63387, at doses up to 100 nmol/kg did not increase serum OT. MK-329, a CCK-A receptor selective antagonist, at a dose of 20 nmol/kg fully inhibited the action of 20 nmol/kg CCK8, while 100 nmol/kg of (R)L-365,260, a CCK-B selective antagonist, had no effect on the CCK8 response. These results, together with previous lesion studies, suggest that vagal CCK-A receptors in the periphery mediate the activation of the oxytocinergic pathway in vivo.

Synthesis and biological activity of CCK heptapeptide analogues. Effects of conformational constraints and standard modifications on receptor subtype selectivity, functional activity in vitro, and appetite suppression in vivo.

A series of modifications of the CCK7 analogue (des-NH2)Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2 was prepared and tested for binding to guinea pig CCK-A and CCK-B receptors and in CCK-A-mediated functional assays. Selected analogues also were tested for appetite suppressant activity in rats. Several conformationally restricted residues in the C-terminal tetrapeptide region, including delta Z-Phe33, (N-Me)Phe33, (N-Me)Asp32, (N-Me)Leu31, and 3PP31 (3PP = trans-3-n-propyl-L- proline) were found to be acceptable modifications at one or both receptor subtypes. The (N-Me)Asp32 and (N-Me)Leu31 modifications afforded potent and selective CCK-A and CCK-B ligands, respectively. SAR studies in the N-terminal acyldipeptide region examined structural requirements for the side chain at position 28, where Gly and Pro replacements were found to possess high affinity at both receptor subtypes. Other conformationally restrictive modifications were less active. All of the analogues that showed high affinity (less than 10 nM) for the CCK-A receptor also were full agonists in amylase release and most were full or nearly full agonists in the phosphoinositide (PI) turnover assay, the most notable exception being the delta Z-Phe33 analogue, which showed 69% of the maximal response in the PI assay. Potent activity in suppression of food intake in rats was found for selected analogues.

Both CCK-A and CCK-B/gastrin receptors mediate pepsinogen release in guinea pig gastric glands.

We evaluated the affinity of cholecystokinin octapeptide (CCK-8), gastrin, and subtype-selective CCK agonists for CCK/gastrin receptors and compared it with the ability of these peptides to stimulate phosphoinositide (PI) hydrolysis and pepsinogen release in guinea pig gastric glands. Competitive binding studies using 125I-labeled Bolton-Hunter-CCK-8 and 125I-gastrin showed the presence of CCK-B/gastrin receptors in gastric glands and dispersed chief cells. In contrast, the potency of peptides in stimulating PI hydrolysis in both gastric glands and dispersed chief cells displayed a profile similar to CCK-A receptors found in pancreatic acini, i.e., CCK-8 = A 71378 greater than A 71623 greater than A 70874 much greater than A 72962 = CCK-8 (desulfated) greater than gastrin II greater than gastrin I. In general, the rank order of potency of peptides for stimulation of PI hydrolysis correlated well with their ability to stimulate pepsinogen release. At concentrations greater than 10 microM, efficacies of gastrin I and II in stimulating pepsinogen release from gastric glands were near 90% of the maximal activity of CCK-8. The inhibitory potency of MK-329, a selective CCK-A receptor antagonist, was similar against either CCK-8 (10 nM) or gastrin I (10 microM), except that a minor portion (approximately 30-40%) of gastrin I-induced pepsinogen release was insensitive to MK-329. The MK-329-insensitive component was inhibited by CI-988, a potent and selective CCK-B/gastrin receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of potent and selective CCK-A receptor agonists from Boc-CCK-4: tetrapeptides containing Lys(N epsilon)-amide residues.

A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N epsilon-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the epsilon-amino group of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists.

Boc-CCK-4 derivatives containing side-chain ureas as potent and selective CCK-a receptor agonists.

Novel Boc-CCK-4 derivatives were communicated recently as having high potency and selectivity for the CCK-A receptor (Shiosaki et al. J. Med. Chem. 1990, 33, 2950-2952). While Boc-CCK-4 binds selectively to the CCK-B receptor, replacement of the methionine with an N epsilon-substituted lysine dramatically reversed receptor selectivity, leading to the development of this novel series of tetrapeptides. A detailed structure-activity analysis of a series of urea-substituted tetrapeptides, represented by the general structure Boc-Trp-Lys(N epsilon-CO-NHR)-Asp-Phe-NH2, revealed that a number of substituted phenyl, naphthyl, and aliphatic urea residues in the lysine side chain yielded potent and selective CCK-A ligands. These tetrapeptides elicit full agonist responses in stimulating pancreatic amylase release that are effectively blocked by a selective CCK-A receptor antagonist. Conversion of the urea to a thiourea significantly reduced CCK-A binding potency as did replacement of the lysine with the homologous ornithine or homolysine. Tetrapeptides that were partial agonists (less than 80% efficacy) in phosphoinositide (PI) hydrolysis relative to CCK-8 did not exhibit high-dose inhibition of amylase secretion in guinea pig acini.

Characterization of two novel cholecystokinin tetrapeptide (30-33) analogues, A-71623 and A-70874, that exhibit high potency and selectivity for cholecystokinin-A receptors.

Based on their relative affinities for cholecystokinin octapeptide (26-33) (CCK-8), cholecystokinin tetrapeptide (30-33) (CCK-4), desulfated CCK-8, and gastrin, cholecystokinin (CCK) receptors have been classified as CCK-A (alimentary) and CCK-B (brain). Selective nonpeptide antagonists of CCK-A and CCK-B receptors, as well as highly selective CCK-A and CCK-B peptide agonists, have been described. We report here the characterization of two novel CCK-4-based peptides, A-71623 and A-70874. In radioligand binding assays, the IC50 values for A-71623 and A-70874 were 3.7 and 4.9 nM in guinea pig pancreas (CCK-A) and 4500 and 710 nM in cerebral cortex (CCK-B), respectively. Both were agonists in stimulating pancreatic amylase release, and their stimulatory effects were potently inhibited by the CCK-A antagonist L-364,718. A-71623 was a full agonist and A-70874 was a partial agonist (approximately 80%) in stimulating phosphoinositide breakdown in pancreas. Both peptides also were potent agonists in stimulating CCK-A receptors in the ileum. They were, however, weak and behaved as partial agonists in calcium studies in NCI-H345 cells, which possess CCK-B/gastrin receptors. In guinea pig gastric glands, the affinities of A-71623 and A-70874 for the CCK-B/gastrin receptor were 11 and 1.6 microM, respectively. These results demonstrate that A-71623 and A-70874 are potent and selective agonists at CCK-A receptors. The preferential interaction of these novel CCK-4 analogs with CCK-A receptors is in contrast to other CCK-4-based peptides, which are primarily selective for CCK-B receptors. In addition, A-71623 and A-70874 are the first two examples of potent CCK-A agonists that do not contain a tyrosine residue whose sulfation is required for potent CCK-A agonist activity of larger peptides.

A71378: a CCK agonist with high potency and selectivity for CCK-A receptors.

Receptors for the brain and gut peptide cholecystokinin (CCK) have been classified into two classes, CCK-A and CCK-B. To date, peptide analogues with selectivity for the CCK-B receptors have been identified, and selective antagonists for CCK-A and CCK-B receptors have been reported as well; until now, there have been no reports of highly selective CCK-A agonists. Herein we describe the properties of A71378 [desamino-Try(SO3H)-Nle-Gly-Trp-Nle-(N-methyl)Asp-Phe-NH2], a highly selective CCK-A receptor ligand. Characterization of A71378 was carried out in the guinea pig pancreas, cortex, gastric gland, and ileum, as well as in NCI-H345 cells. The IC50 values of A71378 for the pancreatic CCK-A, cortical CCK-B, and gastrin receptor were 0.4 nM, 300 nM, and 1,200 nM, respectively. A71378 proved to be a potent agonist in eliciting pancreatic amylase secretion (EC50 = 0.16 nM) and ileal muscle contraction (EC50 = 3.7 nM). In contrast, A71378 was relatively weak (EC50 = 600 nM) in mobilizing intracellular calcium from NCI-H345 cells, which express CCK-B/gastrin receptors. The high potency and selectivity of A71378 for the CCK-A over CCK-B and gastrin receptors is unprecedented among CCK peptides. Studies on CCK-7 analogues indicate that N-methylation of the Asp residue is responsible for the observed selectivity for CCK-A receptors. This discovery of a selective CCK-A agonist should prove valuable for studies aimed at understanding the physiological roles of CCK-A receptors in the brain and periphery.

Pharmacological and mechanistic studies of cholecystokinin-facilitated [3H]dopamine efflux from rat nucleus accumbens.

Cholecystokinin (CCK) is co-localized with dopamine (DA) in mesolimbic neurons of the CNS and appears to selectively regulate the output of this system. In an attempt to characterize the nature of CCK interactions with mesolimbic DA-containing nerve terminals, we have investigated CCK regulation of [3H]DA overflow from rat nucleus accumbens slices. CCK-8 produced a saturable and potent (EC50 = 3 nM) facilitation of KCl (35 mM)-evoked [3H]DA efflux from nucleus accumbens, but failed to significantly alter [3H]DA efflux from striatum: The stimulatory action of CCK-8 was unaffected by the muscarinic antagonist atropine, the opiate antagonist naloxone, or the selective ion channel blockers tetrodotoxin and nifedipine. Pharmacological studies revealed that non-sulfated CCK-8 and CCK-4 (up to low micromolar concentrations) did not facilitate [3H]DA efflux from accumbens slices. Furthermore, the effect of CCK-8 was selectively and potently (IC50 = 300 nM) inhibited by the Type A-selective CCK antagonist CR-1409. Taken together, these results indicate that CCK regulates DA efflux from mesolimbic nerve terminals via a direct presynaptic action on receptors which display a pharmacological profile that is similar to Type A CCK receptors in gastrointestinal tissues.

Cholecystokinin receptors: relationships among phosphoinositide breakdown, amylase release and receptor affinity in pancreas.

The gut hormone cholecystokinin (CCK) octapeptide stimulates the release of amylase from exocrine pancreas, a process believed to be the result of the breakdown of phosphatidylinositol lipids. To examine further the relationship between phosphoinositide (PI) breakdown and amylase release, we have investigated the effect of N-terminally protected CCK C-terminal fragments in these systems using guinea-pig pancreatic lobules. There was a good correlation between the ability of these fragments to elicit amylase release and their potency in enhancing PI breakdown. In general, the EC50 for amylase release is 10-fold lower than the EC50 for PI breakdown. In addition, a good correlation existed between amylase release and the affinity of CCK fragments for [125I]Bolton Hunter-CCK octapeptide binding sites in pancreatic membranes. We also discovered that N-carbobenzyloxy-CCK tetrapeptide was relatively inefficient in causing PI breakdown, but it caused a near maximal stimulation in amylase release. These findings might reflect an amplification mechanism between PI breakdown and amylase release or that N-carbobenzyloxy-CCK tetrapeptide-induced amylase release is independent of PI breakdown.