Introduction

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CCK, a 33 amino acid peptide first isolated from porcine gut, is a member of a family of peptides that together with gastrin share a common carboxyl-terminal pentapeptide amino acid sequence (Mutt and Jorpes, 1968). Both N-terminal extended forms of CCK have been described as well as truncated forms such as CCK₈, pentagastrin and CCK₄ (Dockray et al., 1978; Larsson and Rehfeld, 1979; Reeve et al., 1990). These peptide fragments of CCK have been useful in pharmacologically identifying two receptor types: CCK_A and CCK_B (Innis and Synder, 1980; Grider and Makhlouf, 1987; Hughes et al., 1990; Hill et al., 1987). CCK_A receptors, found primarily in pancreatic acinar cells and in the gastrointestinal tract, have high affinity for sulfated CCK (i.e., CCK8S) and gastrin and a lower affinity for unsulfated CCK₈, pentagastrin and CCK₄. In contrast, the CCK_B receptors that predominate in brain have high affinity for pentagastrin and the tetrapeptide CCK₄ (Hughes et al., 1990). More recently, confirmation of receptor heterogeneity has occurred with the development of selective, high affinity nonpeptide antagonists (Woodruff and Hughes, 1991) and the recent cloning of the CCK_A and CCK_B receptors (Wank et al., 1992a; Ulrich et al., 1993; Wank et al., 1992b; Pisegna et al., 1992; Lee et al., 1993).

The physiological functions described for the CCK_A receptor in the periphery are primarily related to pancreatic secretion of amylase and insulin, although in the gastrointestinal tract pepsinogen is secreted in addition to stimulation of longitudinal smooth muscle (Woodruff and Hughes, 1991). In the central nervous system, CCK_A receptors have been identified in the substantia nigra and striatum and appear to influence activity of the dopaminergic system (van Dijk et al., 1984; Hill et al., 1990). CCK_A receptors have also been found in the dorsal raphe, area postrema, nucleus tractus solitarius and interpeduncular nucleus (Woodruff and Hughes, 1991).

Historically, elucidation of the physiological role of peripherally located CCK_B receptors has been limited. However, with the development of such specific antagonists as CI-988 and L-365,260, the significance of this receptor is becoming more apparent. Several investigators have reported that CCK_B receptors may mediate secretory and contractile responses in the guinea pig (Lucaites et al., 1991, Grider and Makhlouf, 1990). Specific agonists that produce CCK_B-mediated peripheral effects include pentagastrin and CCK₄ (Lucaites et al., 1991).

CCK_B receptors are the predominant CCK-receptor subtype in the brain. Infusion of pentagastrin into the lateral ventricles of sheep has been shown to produce behaviors equated to fear (Della-Fera and Baile, 1979). Hughes et al. (1990) showed that the specific receptor antagonist, CI-988, reduced anxiety in the mouse black-white test and in the marmoset-human threat test. In humans with a history of panic disorder, i.v. administered CCK₄ produces spontaneous panic-like symptoms that are indistinguishable from endogenous panic attacks. These symptoms have been characterized by increases in anxiety and physiological signs such as increased heart rate and blood pressure (Bradwejn et al., 1992). Other investigators have reported that the nonselective CCK_A and CCK_B receptor agonist, CCK_8S, also produces increases or decreases in HR (dose dependent) and increases in mean arterial pressure of rats that are blocked by the CCK_A selective antagonist, devazepide (Guarini et al., 1988; Janssen et al., 1991; Gaw et al., 1995). The purpose of this study is to determine which CCK receptor subtype mediates the cardiovascular responses observed by Bradwejn et al. (1992) after administration of CCK₄.

	Methods

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Compounds

Cholecystokinin 4 (structure H-Trp-Met-Asp-Phe-NH2) was from Bachem Feinchemikalien AG, Germany. The CCK antagonists, CP-212,454 (N-(1-t-butyl) 2-[3-(3-chlorophenylureido)-2-oxo-5-phenyl-8-methyl-2,3,4,5-tetrahydro-1H-(1)benzazepin-1-yl] ethanoic acid amide), CP-310,713 (N-(1-t-Butyl) 2-[3-(3-carboxyphenylureido)-2-oxo-5-cyclohexyl-8-methyl-2,3,4,5-tetrahydro-1H-(1)benzazepin-1-yl] ethanoic acid amide), L-365,260, devazepide and CI-988 were synthesized by Pfizer Inc. (Groton, CT).

Receptor Binding

For CCK_B receptor binding assays guinea pig cortex was homogenized with a Teflon homogenizer in 20 vol of 50 mM Tris HCl (pH 7.4) containing 5 mM MnCl₂ at 4°C and centrifuged at 100,000 × g for 30 min. The supernatant was discarded and the pellet resuspended and spun again. The pellet was diluted to a concentration of 10 mg/ml (original wet weight) with assay buffer (10 mM HEPES, 5 mM MgCl₂, 1 mM EGTA, 130 mM NaCl and 0.2 mg/ml bacitracin, pH 6.5) before use. The incubation reaction was initiated by the addition of 50 µl of tissue to 96-well plates containing 150 µl of assay buffer with 1% DMSO final concentration, 50 pM final concentration of ¹²⁵I-BH-CCK_8S (Du Pont NEN, Boston, MA) and the appropriate concentration of drug or vehicle. Nonspecific binding was estimated using 1 µM CCK_8S. The reaction was terminated by spinning the plates using a H1000B rotor at 3000 rpm for 5 min at 4°C. The pellet was washed with 200 µl of 50 mM Tris HCl and respun. The supernatant was again discarded, the pellet resuspended and the tissue harvested onto Betaplate filters (Wallac Oy, Turku, Finland) soaked in 0.2% polyethylenimine for 2 hr using a Skatron cell harvester (Skatron Instruments, Inc., Sterling, VA). The filtermats were dried and counted on a Betaplate counter (Wallac Inc., Gaithersburg, MD) for 45 sec per sample.

For CCK_A receptor binding the pancreas was dissected from a male Hartley guinea pig and placed in saline. Fatty tissue and blood vessels were dissected away and the tissue placed in 20 vol of buffer (50 mM Tris HCl, pH 7.4, 0.35 mg/ml bacitracin and 0.5 mg/ml soybean trypsin inhibitor) at 4°C and minced using scissors. The tissue was homogenized (Polytron, setting no. 9 for two 15-sec bursts), strained through gauze and centrifuged at 100,000 × g for 15 min at 4°C. The supernatant was discarded and the pellet resuspended in 20 vol of buffer and recentrifuged. The final pellet was diluted to a concentration of 1.25 mg/ml (original wet weight) in buffer and kept on ice until used. The incubation reaction was initiated by the addition of 100 µl of tissue to 96-well plates containing 150 µl of incubation buffer (50 mM Tris HCl, pH 7.4, and at final concentration 5 mM MgCl₂, 5 mM dithiothreitol and 1% DMSO) with 60 pM final concentration of ¹²⁵I-BH-CCK_8S, and drug or vehicle. Nonspecific binding was estimated using 1 µM L-364,718. After a 30-min incubation the reaction was terminated by rapid filtration using a Skatron cell harvester onto GF/B filters that were soaked for 2 hr in 50 mM Tris HCl, 0.1 mg/ml bovine serum albumin. The filtermats were dried and counted on a Betaplate counter for 45 sec per sample.

Phosphatidylinositol Turnover

AR 4-2J rat pancreatoma cells obtained from Dr. J. Putney (NIEHS, Research Triangle Park, NC) were grown in DMEM supplemented with L-glutamine and 10% fetal bovine serum (FBS). AR 4-2J cells were prelabeled with 10 µCi/ml [³H]-myo-inositol overnight. The cells were incubated with agonists for 45 min in the presence of LiCl 10 mM and the reaction terminated by adding CHCl3:MeOH (1:2). The cells were harvested with PBS containing 3 mM EDTA, spun down and resuspended in PBS with 20 mM HEPES and 3 mg/ml D-glucose at a concentration of 1 to 5 × 10⁶ cells/ml. Cells were exposed to antagonists 10 min before agonist exposure. [³H]-inositol phosphates were isolated by a batch technique using a dowex AG1-X8 anion exchange resin. Corrected IC50s (K_i) were calculated by K_i = IC50/1 + [pentagastrin]/[EC₅₀ pentagastrin], where [] = concentration.

Gastric Acid Secretion

Gastric acid secretion studies were conducted in rats using a modification of the pylorus ligation model described by Hakkinen et al. (1991). Fasted male Sprague-Dawley rats (125-250 g) were anesthetized by inhalation with methoxyflurane (Metofane, Pitman-Moore, Inc., Chicago, IL) and the pylorus ligated. Compounds were administered in a vehicle DMSO:emulphor:saline (5:15:80) by s.c. injection (4 ml/kg). Pentagastrin was administered in a 1:99 vehicle of DMSO:saline (v:v). Rats were killed 2 hr after administration of drugs and pentagastrin and the gastric fluid was diluted with water and titrated to pH 7.0 with 0.1 N sodium hydroxide using a Radiometer TTT85 Titrator and an ABU80 Autoburette (Radiometer America, Inc., Westlake, OH). The amount of sodium hydroxide used was taken as a direct measure of the titratable acid (expressed in µEq) in the sample. The acid content was calculated per ml of gastric fluid and normalized to the time of the ligation and the body weight of the rat.

In Vivo Cardiovascular Studies

Animals. All animal studies were conducted in accordance with protocols approved by the Pfizer Institutional Animal Care and Use Committee. Male Hartely guinea pigs were obtained from Charles River Breeding Laboratories (Wilmington, MA). Purpose-bred mongrel dogs were obtained from Hazelton Labs (Kalamazoo, MI). Animals were housed on a 12-hr light cycle (0700-1900 hr) at 27 ± 5°C.

Cardiovascular measurements. Animals were studied in the conscious state. Guinea pigs were placed in Plexiglas restraining tubes. Carotid catheters were connected to a Statham P23ID pressure transducer (Ohmeda, Oxnard, CA) positioned at the level of the heart and interfaced with a Gould (Gould Instrument Systems Inc., Valley View, OH) transducer amplifier (model 20-4615-50). The telemetered signals from dogs were transformed back to calibrated analog signals using a Data Sciences UA10 Universal Adapter D/A converter. The pulsatile waveforms were displayed on an Astromed MT95000 polygraph (Astromed, West Warwick, RI). Mean arterial pressure and heart rate were derived from a beat-to-beat analysis of the pulsatile waveform using Po-Ne-Mah model MA-1 data acquisition and analysis software (Po-Ne-Mah, Inc., Simsbury, CT). Values were averaged over 60-sec intervals during base-line periods. To increase the sensitivity of the system to acute changes in pressure and heart rate, measurements were averaged over 10-sec intervals after i.v. challenge with CCK₄.

Dose response to CCK₄. A dose of CCK₄ that gave a robust and reproducible increase in mean arterial pressure and decrease in heart rate after i.v. bolus administration was established. A dose response curve to CCK₄ was generated over a range of 1 to 160 µg/kg in guinea pigs (n = 4) and 1 to 20 µg/kg in mongrel dogs (n = 3). The peptide was dissolved in 0.9% saline vehicle at a volume of 1 ml/kg i.v. in guinea pigs or 0.1 ml/kg i.v. for dogs. The dose concentrations were delivered in a random fashion. At the end of the study, selected doses were repeated in the animals to evaluate whether there was any tolerance or tachyphylaxis to the CCK₄ injection.

Experimental protocol. After obtaining stable base-line measurements of mean arterial pressure and heart rate, the animal was challenged with an i.v. bolus injection of the 0.9% saline vehicle and changes in pressure and heart rate recorded. Fifteen min later, the maximal control cardiovascular response to an i.v. bolus injection CCK₄ (20 µg/kg for guinea pigs; 10 µg/kg for dogs) was measured. For guinea pig studies, the CCK receptor antagonist or vehicle (3 ml/kg) was administered by a slow i.v. push. For dog experiments, the antagonist or vehicle (2% Tween 80 in water at 4 ml/kg) was administered orally. All antagonists were prepared in a 5:5:90 (v:v:v) DMSO/emulphor/5% dextrose vehicle. CCK₄ challenges were then repeated at 15, 45, 75, 105 and 135 min and the peak changes in mean arterial pressure and heart rate were recorded. The activity of a given CCK receptor antagonist was expressed as its ability to attenuate the cardiovascular response to CCK₄ as compared to the maximal control CCK₄ response recorded initially in a given animal.

Statistical analyses. All results are reported as the mean ± S.E.M. A one-way analysis of variance with an unpaired Student's t test was used for receptor binding and gastric acid secretion studies. When examining the cardiovascular effects of CCK antagonists, a two-way analysis of variance with repeated measures using time and treatment as factors was performed. If the variances were not homogenous, a Student-Newman-Keuls method was used for pairwise multiple comparisons, otherwise a Bonferroni t test was performed. A value of P < .05 was considered significant.

	Results

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Effect of agonists and antagonists on ¹²⁵I-BH-CCK_8S binding to CCK_B and CCK_A receptors. The binding of agonists and antagonists to CCK_B and CCK_A receptors was examined using ¹²⁵I-BH-CCK_8S binding to guinea pig cortex and pancreas, respectively. Of the antagonists tested, CP-310,713 had the highest affinity and selectivity for CCK_B receptors followed by CI-988, CP-212,454 and L-365,260 (table 1). Devazepide was 500-fold selective for CCK_A receptors, binding with an IC₅₀ value of 0.23 ± 0.015 nM.

Effect of antagonists on pentagastrin-induced phosphatidylinositol turnover in rat AR 4-2J pancreatoma cells. Pentagastrin (PG) stimulated PI turnover in late passage AR 4-2J rat pancreatoma cells with an EC₅₀ of 0.3 nM. The specific CCK_B receptor antagonists, L-365,260, CI-988, CP-212,454 and CP-310,713 all showed dose-dependent inhibition of the pentagastrin-induced (0.33 nM) PI turnover with K_i values of 4.1, 2.4, 0.65 and 0.23 nM, respectively. In contrast, the selective CCK_A receptor antagonist, devazepide, had little effect on pentagastrin-induced PI turnover with a K_i value >300 nM (table 2).

Effect of antagonists on pentagastrin-induced acid secretion in rats. The specific CCK_B receptor antagonists, L-365,260, CP-212,454 and CP-310,713 all showed dose-dependent inhibition of gastric acid secretion with s.c. ID₅₀ values of 1.5, 0.80 and 0.01 mg/kg, respectively. The CCK_B receptor antagonist, CI-988, blocked 60% of the pentagastrin-mediated effect with an ID₅₀ value of 0.08 mg/kg, consistent with some reports indicating that it is a partial agonist at this receptor. The CCK_A receptor antagonist, devazepide, was the least potent with an ID₅₀ of 8.0 mg/kg when given s.c. (fig. 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Dose-response curves for the CCKA receptor antagonist, devazepide (), and the CCKB receptor antagonists L-365,260 (), CP-212,454 () and CI-988 (), to antagonize pentagastrin-induced acid secretion in the pylorus ligated rat. Data are expressed as percentage of control maximum response to pentagastrin and each point represents the mean of not less than 10 rats. The coefficient of variation for each point ranged from 5 to 25%.

Effect of CCK₄ on heart rate and mean arterial pressure. The cardiovascular effects of CCK₄ on HR and MAP were examined in the conscious guinea pig and dog. Dose-response curves in guinea pigs with i.v. bolus doses of CCK₄ from 1 to 160 µg/kg were generated for changes in HR and MAP. Decreases in HR and increases in MAP were observed in a dose-dependent manner up to 20 µg/kg of CCK₄. Doses of CCK₄ between 20 and 160 µg/kg produced only small increases in the magnitude of both the HR and MAP responses. No tachyphylaxis was observed when CCK₄ was repeatedly administered every 30 min over a 135-min period (fig. 2). Because 20 µg/kg of CCK₄ produced the most consistent response, this dose was chosen for all subsequent studies to compare receptor antagonists in the guinea pig.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Effect of repeated doses of CCK4 (20 µg/kg, i.v.) on blood pressure in the conscious guinea pig. CCK4 was administered at 30-min intervals for 135 min starting 15 min before administration of the CCK4 antagonist, CP-212,454 or vehicle.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of the CCKB antagonist on heart rate changes in the conscious dog. Top panel, Effect of 50 mg/kg of orally administered CP-212,454 () or 4 ml/kg vehicle () on basal heart rate before CCK4 challenges. Bottom panel, Percentage change in maximal heart rate response to CCK4 after CP-212,454 () or vehicle (). Each point represents the mean of at least four dogs.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of the CCKB antagonist on mean arterial blood pressure changes in the conscious dog. Top panel, Effect of 50 mg/kg of orally administered CP-212,454 () or 4 ml/kg vehicle () on basal mean arterial blood pressure before CCK4 challenges. Bottom panel, Percentage change in maximal mean arterial pressure increase to CCK4 after CP-212,454 () or vehicle (). Each point represents the mean (±S.E.M.) of at least four dogs.

Dose-response effect of CCK_A and CCK_B receptor antagonists on HR and MAP. Dose-response curves for changes in HR and MAP with specific CCK_A and CCK_B receptor antagonists in guinea pigs are shown in figures 3 and 4, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Dose-response curves for the CCKA receptor antagonist, devazepide (), and the CCKB receptor antagonists L-365,260 (), CP-212,454 (), CI-988 (), CP-310,713 () to antagonize CCK4-induced decreases in HR in the guinea pig. Data are expressed as percentage of control maximum response to CCK4 and each point represents the mean (±S.E.M.) of not less than four guinea pigs.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Dose-response curves for the CCKA receptor antagonist, devazepide (), and the CCKB receptor antagonists L-365,260 (), CP-212,454 (), CI-988 (), CP-310,713 () to antagonize CCK4-induced increases in MAP in the guinea pig. Data are expressed as percentage of control maximum response to CCK4 and each point represents the mean (±S.E.M.) of not less than four guinea pigs.

	Discussion

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Our study demonstrates that the cardiovascular responses to CCK₄ in the guinea pig and dog are mediated by the CCK_B receptor subtype. Three established models, in vitro receptor binding, inhibition of pentagastrin stimulated phosphatidylinositol hydrolysis in AR 4-2J cells and in vivo inhibition of pentagastrin-induced acid secretion, were used to rank order the affinity of antagonists for the CCK_B receptor. The rank order of potency of antagonists in blocking CCK₄-induced HR and MAP changes in the guinea pig corresponded closely to the rank order of their affinity and/or activity in all three models. The qualitative changes induced by CCK₄ on HR, but not MAP, appear to be species dependent. CCK₄ decreased the HR in the guinea pig although in the dog an increase was observed. However, in the rat, administration of CCK_8S has been reported to increase HR at low doses but at higher doses there is a transient decrease followed by an increase in HR (Janssen et al., 1991). MAP is consistently increased by CCK in studies performed in our laboratory using guinea pigs and dogs and by other investigators using rats (Gaw et al., 1995).

The anxiolytic activity of CCK receptor antagonists has been shown to be mediated through the CCK_B-receptor subtype (Harro and Vasar, 1991; Singh et al., 1991). Bradwejn et al. (1992) has shown that the anxiogenic effects of increasing i.v. doses of CCK₄ in patients with panic disorders showed a strong relationship to the increases in HR and diastolic blood pressure experienced in the same patients. The central or peripheral origin of the cardiovascular activity of CCK in animals is uncertain. No definitive study demonstrating the partitioning of CCK into the brain for central activity after i.v. administrations has been reported. However, high immunoreactive concentrations of the endogenous peptide have been found in regions of the brain associated with cardiovascular control such as the nucleus tractus solitarius (NTS) and area postrema (AP) (Newton and Maley, 1985; Howes et al., 1989). These regions can also be indirectly affected by stimulation of CCK at peripheral vagal afferent fibers leading into this area of the brain (Koyama et al., 1990). This hypothesis is supported by experiments where i.v. injection of CCK has been shown to stimulate gene expression of Fos-like protein, an indicator of neural stimuli, not only in the nucleus tractus solitarius and area postrema, but also in the ventrolateral medulla that receives projections from the nucleus tractus solitarius (Luckman, 1992).

Although the cardiovascular activity of different CCK peptides has been known for some time, the association to a particular receptor is just beginning to be addressed. In 1991, Mei and Han described that intrathecal CCK₈ can antagonize the hypotension induced by µ and  opioid agonists. The antagonist activity was mediated by the CCK_B receptor subtype (Mei and Han, 1993), as evidenced by the 20- to 40-fold greater potency of the CCK_B receptor antagonist, L-365,260, over the CCK_A receptor antagonist, devazepide, to block the effect of CCK₈. More recently, Gaw et al. (1995) concluded that the cardiovascular effects of CCK_8S could be attributed to CCK_A-mediated activity. This conclusion was supported by data that showed bolus i.v. doses of CCK_8S in the pithed rat produced increases in the MAP and decreases HR. These effects curiously were only partially attenuated with devazepide but not at all with L-365,260 or CI-988. However, in the same report when CCK₄ was used, similar increases in MAP were produced and were unaffected by treatment with devazepide, L-365,260 or phentolamine. Because CCK_8S has almost equal affinity to both the CCK_A and CCK_B receptors (see table 1), the unaccounted for activity observed after devazepide with CCK_8S may be due to the remaining CCK_B activity. Gaw et al. (1995) also concluded that because neither devazepide nor L-365,260 blocked the increase in MAP induced by CCK₄, these effects were not mediated by either CCK_A or CCK_B receptors. Our data suggest that even though L-365,260 has affinity for CCK_B in vitro, it is a relatively weak antagonist for CCK_B-mediated activity in all three of our functional assays. In the binding assay, the rank order for potency at displacing ¹²⁵I-BH-CCK_8S binding for the CCK_B receptor was CP-310,713 > CI-988 > CP-212,454 > L-365,260 > devazepide. The order of potency was closely followed for inhibiting pentagastrin-stimulated PI hydrolysis in rat pancreatoma cells and inhibition of pentagastrin-induced acid secretion in the rat model that also followed the observed activity in the guinea pig cardiovascular system.

The contribution of CCK to endogenous tone of the cardiovascular system appears to be neglible under normal circumstances. Figures 7 and 8 show that when 1.0 µM/kg of CP-212,454 is given as a bolus i.v. dose that concentrations are sufficient to antagonize the transient increases in HR and MAP for at least 135 min yet no changes were observed in the basal values at each time period before CCK₄ challenges. At the highest dose of CP-212,454, there was a significant decrease in basal HR with no concomitant drop in MAP. The decrease in HR was not blocked by a 0.1 mg/kg i.v. atropine pretreatment (data not shown) indicating that the effect was not vagally mediated. Two other possibilities are a direct negative chronotropic effect on the heart or a decrease in sympathetic tone. In the experimental rat model of hemorrhagic shock, Guarini et al. (1988) has shown that the CCK_8S quickly produces a sustained elevation in blood pressure and pulse amplitude that is significantly antagonized by sympatholytics such as prazosin, reserpine and yohimbine. Interestingly, in this study devazepide, given i.v. at 0.01 to 0.05 mg/kg (0.025-0.123 µmol/kg) but not intracerebroventricularly (0.002 mg/kg or 0.005 µmol/kg), completely antagonized the response of CCK_8S. Although these and other data by Gaw et al. (1995) indicate a sympathetic component for the effect of CCK_8S but not CCK₄, the issue of whether the activity is of central or peripheral origin still remains unclear because a specific CCK_B receptor antagonist has not been examined centrally.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Effect of the CCKB antagonist on HR changes in the conscious guinea pig. Top panel, Effect of 0.1 µM/kg (), 1.0 µM/kg (), 10 µM/kg () or 3 ml/kg vehicle () of CP-212,454 (i.v.) on HR changes induced by CCK4 (20 µg/kg, i.v.) challenges. Bottom panel, Percentage change in maximal heart rate decrease to CCK4 after 0.1 µM/kg (), 1.0 µM/kg (), 10 µM/kg () or 3 ml/kg vehicle () of CP-212,454 (i.v.). Each point represents the mean (±S.E.M.) of at least five guinea pigs.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Effect of the CCKB antagonist on mean arterial blood pressure changes in the conscious guinea pig. Top panel, Effect of 0.1 µM/kg (), 1.0 µM/kg (), 10 µM/kg () or 3 ml/kg vehicle () of CP-212,454 (i.v.) on mean arterial pressure changes induced by CCK4 (20 µg/kg, i.v.) challenges. Bottom panel, Percentage change in maximal mean arterial pressure increase to CCK4 after 0.1 µM/kg (), 1.0 µM/kg (), 10 µM/kg () or 3 ml/kg vehicle () of CP-212,454 (i.v.). Each point represents the mean (±S.E.M.) of at least five guinea pigs.

Our data from the dog and the guinea pig suggest that CCK₄-induced cardiovascular changes are mediated by the CCK_B receptors subtype. This is consistent with reports that a CCK_B receptor antagonist can block the cardiovascular responses in man elicited by CCK₄ or pentagastrin (Bradwejn et al., 1992). One explanation for the conflicting observations in the literature would be that the CCK_A receptors may reside on peripheral afferent fibers (Luckman, 1992) that when stimulated by CCK_8S modulate sympathetic outflow from the brain resulting in increases in HR and blood pressure that are blocked with prazosin or devazepide (Guarini et al., 1988). A separate population of peripheral CCK_B may be more directly involved in modulating cardiovascular responses not influenced by the sympathetic system (Gaw et al., 1995). Therefore these effects were previously indistinguishable with the nonspecific agonist, CCK_8S, but can be separated using the selective, peripheral CCK_B agonist, CCK₄.