Dissolving microneedle based transdermal delivery of therapeutic peptide analogues.

Microneedle technology offers a viable means of delivering biologically active pharmaceutical agents across the skin in a minimally invasive and virtually pain free manner. Previous work detailed the first successful transdermal delivery of a model peptide drug, polymyxin b, utilising a dissolving polymer-based microneedle system. The focus of this study was to examine the ability of a dissolving microneedle system to deliver a range of peptides of different sizes and properties. Analogue versions of 2 existing therapeutic peptides; pentagastrin and sincalide, were synthesised utilising Fmoc based solid phase peptide synthesis (SPPS) chemistry techniques and once successfully synthesised and purified, the peptide analogues were characterised using LC-MS. The peptide analogues were then incorporated into PVP/trehalose microneedle formulations. Skin permeation testing, in addition to skin penetration testing, was carried out to determine the effectiveness of the microneedle system to deliver the peptide analogues through porcine skin. The results obtained from these studies were then compared with the permeation results obtained utilising polymyxin B as the peptide drug cargo to evaluate the PVP/trehalose microneedle system's suitability to successfully deliver therapeutic peptides. Results indicated that the microneedle system successfully systemically delivered a higher overall percentage of the encapsulated peptides at an initially faster rate than peptide loaded control discs and in therapeutically relevant concentrations.

Characterization of the GI transit conditions in Beagle dogs with a telemetric motility capsule.

In preclinical research, Beagle dogs are an important model for formulation development and for evaluation of food effects on drug absorption. In this study, the gastrointestinal transit conditions in Beagle dogs were studied with a telemetric motility capsule at different intake conditions. In a cross-over study design, the SmartPill® was given to six Beagle dogs to measure transit times, pH values, pressures and temperatures in the different parts of the canine GI tract. Moreover, the effects of commonly applied pre-treatments as with pentagastrin and famotidine on GI transit conditions were investigated. The gastric transit time in fasted state was short (0.57 ±â€¯0.37 h) and only slightly affected by the pre-treatments. In fed state, gastric transit was clearly prolonged (2.94 ±â€¯0.91 h). The mean intestinal transit time was in the range of 1-2 h and not affected by the intake conditions. The gastric pH values in fasted and fed Beagle dogs were highly variable, but pre-treatment with pentagastrin and famotidine clearly decreased variability. Pre-treatment with pentagastrin resulted in minimum pH values around 0.5 pH units lower than without pre-treatment. Oral administration of famotidine led to constantly elevated pH values of pH 7-8. The maximum pressures in the canine GI tract did not vary significantly between the study arms and typically, maximum pressures of up to 800 mbar were observed in the stomach. The comparison of the data from this study with recent SmartPill® data from humans revealed that major differences could be observed with respect to gastric transit times in fed state, small intestinal transit times as well as maximum pressures arising during GI transit. These differences should be kept in mind if the dog model is used to assess the in vivo performance of solid oral dosage forms intended for use in humans.

Modeling the development of panic disorder with interoceptive conditioning.

Panic disorder is characterized by the paroxysmal occurrence and fear of bodily symptoms. In recent years it has been proposed that patients "learn" to fear cardiorespiratory sensations through interoceptive conditioning. This study sought to model the initial stage of this process in healthy volunteers (N=44) using mild cardiac sensations. An additional aim was to explore whether anxiety sensitivity - a known risk factor for panic disorder - modulates such interoceptive learning. Infusions of pentagastrin and saline were used to manipulate the presence versus absence of cardiac sensations, respectively, and served as conditioned stimuli in a differential interoceptive conditioning paradigm. Inhalation of 35% CO2-enriched air served as the panicogenic, unconditioned stimulus (UCS). In half of the participants ("prepared" condition), cardiac sensations caused by pentagastrin were followed by inhalation of CO2-enriched air (penta CS+), whereas the absence of such sensations (saline) was followed by room air (saline CS-). The reversed combination ("unprepared" condition) was used in the other half of the participants. Conditioning effects showed up for self-reported UCS-expectancy, but not for skin conductance and anxiety ratings. Only participants from the prepared group learned to expect the UCS, and differential learning was impaired with higher scores on anxiety sensitivity. Expectancy learning was more easily established towards the presence compared to the absence of cardiac sensations, whereas the reverse effect was observed for safety learning. Modeling impaired discriminatory learning and the moderating effect of anxiety sensitivity provides new insight in the development of panic disorder.

Comparative assessment of calcitonin stimulation test using calcium gluconate and pentagastrin and the usefulness of procalcitonin basic and post-stimulation concentrations in the diagnosis of patients after surgery for medullary.

OBJECTIVES: The aim of the study was to compare the calcitonin (CT) stimulation tests with tests of calcium gluconate (CaG) and pentagastrin (PG), their tolerance and usefulness of PCT in the patients' diagnosis with active Medullary thyroid cancer (MCT) after thyroidectomy. METHODS: CT was marked in serum by the immunosorbent sandwich test. PCT was marked by the immunosorbent sandwich test, with the final reading of fluorenscence. PG was given intravenously at a dose of 0.5 mg/kg body weight for 10 seconds. CaG was also given by intravenous injection at a dose of 2.5 mg of elemental Ca/kg body weight at a rate of 5ml/min, for minimum 3 minutes. Blood was taken at the 0 minute, the 3 and 5 minute after getting the stimulating substances. RESULTS: The post-stimulation CT concentration in the 3 and 5 minute of the CaG test vs PG is significantly higher compared to the baseline. The maximal stimulation of the CT is in the 3 minute, but higher concentrations occurred using the CaG. CONCLUSION: The results of the study suggest a similar diagnostic value of the tests with CaG compared to the PG as stimulants. In the present study we noticed a trend of basic and post-stimulation concentrations of PCT to increase in the tests with PG and CaG which correspond with the elevated concentrations of CT.

Low-dose calcium versus pentagastrin for stimulation of calcitonin in chronic hemodialysis patients: a pilot study.

CONTEXT: Elevated calcitonin levels occur in up to 46% of patients with chronic hemodialysis (CHD) and frequently reflect benign C-cell hyperplasia rather than medullary thyroid carcinoma. For the differential diagnosis of hypercalcitoninemia, the pentagastrin-stimulated calcitonin test was used until its availability became restricted. OBJECTIVE: This study sought to compare calcium and pentagastrin in terms of their ability to stimulate calcitonin secretion and their side effects in patients with CHD. SETTING AND DESIGN: This prospective pilot study was conducted at the chronic hemodialysis unit of the Medical University of Vienna between December 2012 and September 2013. PATIENTS: We studied six male patients with CHD with elevated basal calcitonin levels. INTERVENTION: The stimulation test was performed first with 0.5 µg/kg pentagastrin and then with 1 mg/kg calcium after a median washout period of 7 (6-9) months. MAIN OUTCOME MEASURES: We measured calcitonin, serum ionized calcium, intact PTH (iPTH), and C-terminal fibroblast growth factor 23 levels before and 2, 5, and 10 minutes after iv infusion of the stimulant and assessed the tolerability of the two substances by a questionnaire. RESULTS: Both pentagastrin and calcium significantly stimulated calcitonin secretion at 2 and 5 minutes. Partial correlation analysis revealed a strong association between calcium- and pentagastrin-stimulated calcitonin levels (r=0.875, P < .0001). Only after calcium infusion serum ionized calcium levels increased from 1.09 (0.91-1.16) mmol/l to 1.4 (1.14-1.65) mmol/l at 2 minutes (P < .01) but returned to baseline levels at 5 minutes. Moreover, calcium infusion led to a significant decrease in iPTH levels from 315 (203-723) pg/ml to 182 (121-415) pg/ml at 5 minutes (P < .05) and 171 (91-346) pg/ml at 10 minutes (P < .001). In general, calcium caused fewer and less severe side effects than pentagastrin. CONCLUSIONS: In patients with CHD, the response of calcitonin to calcium and pentagastrin was comparable, making calcium a potential substitute for pentagastrin in these patients.

Use of the pentagastrin dog model to explore the food effects on formulations in early drug development.

The ability to extrapolate dosage performance from in vitro to in vivo situations has an important role in early drug development. In parallel, the Beagle dog has come to represent a useful animal model for extrapolation to humans especially when drugs formulated for humans are to be tested. In this article, the pentagastrin-induced Beagle dog model was validated internally to show that in the colony the dogs were generally responsive to known doses of pentagastrin that produces effects similar to gastrin in the stomach, i.e., increasing gastric acid production and lowering gastric pH. The effect was observed with a short time course, maximum pH lowering was observed between 0.5 and 1h with return to baseline at 2-4h. The dog stomach pH is a better representative of the human fasted stomach with this pretreatment. The ultimate goal was to use these animals in a food effect studies to predict the behavior of formulations in humans. The results for 4 compounds were provided in the dog and a clear relationship between the effect of food in the dog and the effect of food in humans was observed. While the directionality (positive or negative) of the effect could be adequately predicted, the extent of the effect could not be predicted for all the tested formulations of the 4 compounds. The data will be used to generate a database of known compounds from which a correlation can be drawn to make future predictions using the pentagastrin dog model.

Investigation of the rat model for preclinical evaluation of pH-dependent oral absorption in humans.

Many pharmaceutically active compounds are weak electrolytes and are ionizable in the pH range experienced throughout the gastrointestinal tract. Changes in protonation state due to pH changes in the gut can have dramatic effects on solubility, dissolution, and permeation through biological barriers. Preclinical assessment of the pH-dependence of oral absorption is critical for compounds possessing pH-dependent solubility. Here we examine pH-dependent solubility and oral exposure in rat for three model compounds, dasatinib, ketoconazole, and mefenamic acid. Dasatinib and ketoconazole are both weak bases, while mefenamic acid is a carboxylic acid. The effects of gastric pH modulators, pentagastrin and famotidine, were investigated in rat PK studies to assess the applicability of using the rat to evaluate the risk of pH-dependent oral exposure for ionizable compounds. Dasatinib showed similar exposure between control and pentagastrin-pretreated groups, and 4.5-fold lower AUC in famotidine-pretreated rats. Ketoconazole showed a 2-fold increase in AUC in pentagastrin-treated rats relative to control, and 4.5-fold lower AUC in famotidine treated rats, relative to the pentagastrin group. Mefenamic acid showed highly similar exposures among control, pentagastrin-pretreated, and famotidine-pretreated groups. The rat model was shown to be useful for compounds displaying pH-dependent solubility and oral absorption that may be affected by gastric pH modulators.

Gastric acid secretion in experimental acute uremia.

This study was conducted to evaluate gastric acid secretion in acute renal failure, highlighting the roles of renal mass and gastrin hormone. Acute uremic rats were divided into bilateral nephrectomized and bilateral ureteric obstruction groups. Gastric juice was collected for 2 h and analyzed for volume, free acidity, total acidity, and total acid output. Plasma levels of creatinine, urea, and gastrin were also determined. Bilateral nephrectomized and bilateral ureteric obstruction groups showed a significant increase in levels of free acidity, total acidity, and plasma gastrin. Compared with the ureteric obstruction group, nephrectomized rats showed a significant increase in gastric juice volume, total acid output, and plasma gastrin levels. Following pentagastrin stimulation, gastric juice volume, total acid output, free acidity, and total acidity were increased in the bilateral nephrectomy and ureteric obstruction groups compared with the respective control groups. The free and total acidity and total acid output also increased compared with the respective non-stimulated groups. Plasma creatinine and urea levels were significantly positively correlated with plasma gastrin, free acidity, and total acidity. Creatinine was positively correlated with total acid output, and gastrin was positively correlated with total acidity. In conclusion, acute renal failure promotes gastric acid hypersecretion that could potentially be attributed to high levels of gastrin hormone and uremic state per se.

Comparison of serum calcitonin and procalcitonin in detecting medullary thyroid carcinoma among patients with thyroid nodules.

BACKGROUND: To prospectively evaluate the role of procalcitonin (PCT) in detecting or excluding medullary thyroid carcinoma (MTC) among patients with thyroid nodules and increased calcitonin (CT) levels. METHODS: Fourteen of 1236 patients referred for thyroid nodules had increased serum CT >10 pg/mL. A stimulation test with pentagastrin was done and both CT and PCT were measured after stimulation. All patients underwent thyroid ultrasound, fine-needle cytology and, if indicated, surgery with histological and immunohistochemical examination of the surgical specimens. RESULTS: After follow-up, two MTCs were found. These two patients had basal CT >100 pg/mL and detectable (>0.1 ng/mL) PCT, with 100% sensitivity. Pentagastrin stimulated CT achieved values above 100 pg/mL in two MTCs and in other two cases with no MTC outcome (50% PPV and 83% NPV). On the contrary, all patients with no MTC had both basal and stimulated undetectable PCT (100% PPV and 100% NPV). CONCLUSIONS: The addition of basal PCT measurement in patients with thyroid nodule(s) and increased CT may significantly improve accuracy of CT measurement without needing a PG stimulation test.

Development of a canine model to enable the preclinical assessment of pH-dependent absorption of test compounds.

A preclinical canine model capable of predicting a compound's potential for pH-dependent absorption in humans was developed. This involved the surgical insertion of a gastrostomy feeding tube into the stomach of a beagle dog. The tube was sutured in position to allow frequent withdrawal of gastric fluid for pH measurement. Therefore, it was possible to measure pH in the stomach and assess the effect of gastric pH-modifying agents on the absorption of various test compounds. Fasted gastric pH in the dog showed considerable inter- and intra-animal variability. Pretreatment of pentagastrin (6 µg/kg intramuscularly) 20 min prior to test compound administration was determined to be adequate for simulating fasting stomach pH in humans. Pretreatment with famotidine [40 mg orally] 1 h prior to test compound administration was determined to be adequate for simulating human gastric pH when acid-reducing agents are coadministered. Pentagastrin and famotidine pretreatments were used to test two discovery compounds and distinct differences in their potential for pH-dependent absorption were observed. The model described herein can be used preclinically to screen out compounds, differentiate compounds, and support the assessment of various formulation- and prodrug-based strategies to mitigate the pH effect.

Modification of gastric pH in the fasted dog.

OBJECTIVES: The aim was to compare the ability of pretreatments to consistently adjust gastric conditions to low or high pH in the fasted state in dogs. METHODS: Four male Labrador/Labrador-cross dogs weighing 25-35 kg were surgically equipped with a ventricle fistula cannula in the stomach and a jejunal nipple valve stoma. Dogs were fasted overnight before the experiments, with free access to water. The pH in the dogs' stomach was modified either orally with buffers (0.1 mol/l HCl-KCl, 0.05 mol/l glycine-HCl, 0.1 mol/l citrate or 0.1 mol/l BIS-TRIS) or intravenously with pharmacological agents (pentagastrin 4-6 microg/kg, ranitidine 50 mg or omeprazole 1 mg/kg). Intragastric pH was recorded continuously for 2 h with an electrode connected to an ambulatory pH meter. Chyme was collected simultaneously from the jejunal stoma as an approximate measure of gastric emptying. KEY FINDINGS: 0.1 mol/l HCl-KCl buffer p.o. and 1 mg/kg omeprazole i.v. attained low and high gastric pH more reproducibly (11/11 and 6/7 experiments met target values of pH < 3 and > 4, respectively) and for a longer duration (average time exceeding target value 90 and 103 min, respectively) than the other buffers and pharmacological pretreatments. The starting pH did not alter the modifiers' capacity to increase or decrease the pH. However, the lag time before chyme appeared at the jejunal stoma appeared to be longer when the pH was low and shorter when the pH was high. CONCLUSIONS: To achieve a consistently low gastric pH in fasting dogs, 0.1 mol/l HCl-KCl buffer should be administered orally, 15 min before the dosage form. To elevate the gastric pH reproducibly, omeprazole 1 mg/kg should be administered intravenously at least 90 min before oral administration of the dosage form.

Effect of free dietary glutamate on gastric secretion in dogs.

The amino acid, L-glutamate, which is abundant in many foodstuffs, is a potent stimulator of gastric vagal afferents. The aim of the study was to evaluate a role of dietary glutamate in neuroendocrine control of gastric secretion of acid, pepsinogen, and fluid. In mongrel dogs with small gastric pouches surgically prepared according to Pavlov (vagally innervated) or Heidenhain (vagally decentralized), secretion in a pouch was induced by infusion into the main stomach of an amino acid-rich diet lacking glutamate (Elental) or the same diet supplemented with monosodium glutamate (MSG). Having no effect alone, MSG (100 mM) potentiated secretion induced by Elental both in Pavlov and Heidenhain models. In the Pavlov pouch, the effect of MSG was markedly reduced after i.v. injection of granisetron, an antagonist of 5-HT(3) receptors. In the Heidenhain model, MSG enhanced the stimulatory effect of pentagastrin (1 microg/kg, s.c.). In conclusion, dietary glutamate at doses not exceeding its common concentrations in foods substantially potentiates gastric phase secretion induced by stimulation of gastric mucosa with an amino acid-rich diet or by administration of pentagastrin. The effect of glutamate is partially mediated via serotonin secretion and stimulation of 5-HT(3) receptors.

Validation of a conscious rat model for the discovery of novel agents that inhibit gastric acid secretion.

Identification of novel drug molecules requires the extensive evaluation in vitro and in vivo. Following in vitro evaluation it is necessary to efficiently screen numerous novel molecules in vivo using relatively simple methodology that requires small numbers of animals, is rapid to perform, and provides results that can definitely discriminate potential candidates for further investigation. Herein, we describe the results of three standard compounds (omeprazole, a proton pump inhibitor; cimetidine, an histamine H(2) receptor antagonist; and AR-H047108, a potassium competitive acid blocker) in the rat aspiration model (under both basal and stimulated conditions), and compared the effects with those in the pyloric ligation model with a view to comparing the results in terms of sensitivity, robustness and simplicity of the methodology. In the aspiration model, drug or vehicle was administered orally 1 h prior to administration of pentagastrin or dimaprit. Ten minutes later 0.9% NaCl was administered orally and immediately recovered by aspiration. In the pyloric ligation model, drugs or vehicle were administered orally 2 h before ligation in a volume of 10 ml/kg. For each model, the volume of each sample was measured and the acidity was determined. In the aspiration model under basal acid secretion or following stimulation with pentagastrin omeprazole, cimetidine and AR-H047108 produced dose related inhibition of acidity. Omeprazole and cimetidine inhibited acid secretion following stimulation with dimaprit. In the pyloric ligation model omeprazole, cimetidine and AR-H047108 inhibited acid secretion. The profile of each of 3 inhibitors of acid secretion exhibited similar effects irrespective of the degree of stimulation (dimaprit, pentagastrin or pyloric ligation). Thus, based on these robust effects and ease of methodology we would recommend the use of the rat aspiration model with pentagastrin stimulation of gastric acid secretion as the primary in vivo methodology to screen novel inhibitors of acid secretion.

Symptomatic response to blocked and unblocked pentagastrin stimulation in functional dyspepsia. Comparison of responders and non-responders to omeprazole identified in a single-subject trial model.

BACKGROUND: The role of acid in functional dyspepsia is controversial and drug treatment trials indicate that only a subset of patients has acid-related symptoms. A novel single-subject trial design, the Random Starting Day trial (RSD trial), was developed to identify acid-related symptoms. We hypothesized that RSD trial responders and non-responders would react differently to gastric acid stimulation. Development of epigastric pain was expected in RSD trial responders after pentagastrin stimulation during placebo treatment, but not during omeprazole treatment. In non-responders, epigastric pain was expected not to be influenced by gastric acid stimulation or type of treatment. METHODS: Nineteen patients were evaluated. Symptomatic response to pentagastrin (6 microg/kg) was assessed twice in each patient following placebo and omeprazole (40 mg bid) treatment in a randomized, double-blind, cross-over design. Epigastric pain was assessed every 15 for 90 min after stimulation using a 5-graded Likert scale and a VAS scale. A positive acid provocation test was defined as an increase of the Likert score of epigastric pain by at least one grade after pentagastrin stimulation during placebo treatment but not during omeprazole treatment. RESULTS: The acid provocation test was positive in 43% (3/7) of responders compared to only 17% (2/12) non-responders. VAS-score changes showed trends towards a more pronounced symptom reduction during omeprazole treatment in responders compared to non-responders. CONCLUSION: Patients identified as having acid-related dyspepsia more often developed epigastric pain following acid stimulation during placebo and not during omeprazole treatment. Due to the low sample size the difference was not considered significant.

Effect of time of preparation on pentagastrin-induced symptom, endocrine and cardiovascular responses.

Pentagastrin is a cholecystokinin (CCK)-B agonist and laboratory panicogenic agent that produces endocrine (ACTH and cortisol), symptom (anxiety, panic) and cardiovascular (heart rate) responses. Although in vitro data have supported its chemical stability, preliminary data suggested that increasing time between drug preparation and drug infusion could reduce the magnitude of endocrine and symptom responses. The current study examined this possibility. Twenty-one healthy subjects presented at the University of Michigan General Clinical Research Center (GCRC) and had an intravenous catheter inserted. Heart rate, cortisol levels and subjective anxiety were measured before and after pentagastrin and placebo injections. Pentagastrin was prepared either within 60 min of IV infusion (Normal Preparation group) or at least 3.5 h prior to infusion (Early Preparation group). Relative to the Normal Preparation group, Early Preparation subjects had similar heart rate responses but significantly smaller cortisol and subjective anxiety responses. Early preparation of pentagastrin thus appears to weaken endocrine and subjective anxiety responses, highlighting the importance of attending to often overlooked procedural variables (e.g., time between preparation and administration) in studies of this type. The sensitivity of cortisol and anxiety responses to preparation time, but insensitivity of heart rate, is consistent with previous studies suggesting different thresholds of activation for the three response modalities. These differential sensitivities may suggest different and separable CCK-B stimulated pathways for each response, which combine to produce panic, rather than a single, unified CCK-B mediated panicogenic response.

Neuronal excitability in the periaqueductal grey matter during the estrous cycle in female Wistar rats.

Extracellular recordings were made from output neurons in the dorsal half of the periaqueductal gray matter (dPAG) in urethane-anesthetized female Wistar rats. All the neurons were quiescent. A basal level of firing was therefore induced by continuous iontophoretic application of D,L-homocysteic acid (DLH). In the presence of the GABA(A) receptor antagonist bicuculline methiodide (BIC 0-30 nA) the DLH-induced firing increased further, revealing the presence of ongoing GABAergic inhibitory tone on the recorded neurons. The BIC-induced increase in firing rate was significantly greater in neurons recorded during estrus (Est) and late diestrus (LD) compared with proestrus (Pro) and early diestrus (ED) suggesting that GABAergic tone was lower in Est and LD. I.v. injection of the panicogenic cholecystokinin (CCK)(B) receptor agonist pentagastrin (PG, 40 microg kg(-1)) produced an increase in firing rate in 12/17 (70%) of neurons tested in the dPAG. Iontophoretic application of PG (10-30 nA) also produced a current-related increase in firing rate in 73.6% of the neurons tested. The excitatory response was reduced during application of the selective CCK(B) receptor antagonist beta-[2-([2-(8-azaspiro[4.5]dec-8-ylcarbonyl)-4,6-dimethylphenyl]amino)-2-oxoethyl]-(R)-napthalenepropanoic acid (CR2945) (60 nA, n=6). The PG-evoked increase in firing rate was significantly greater in neurons recorded during Est and LD compared with during Pro and ED. Juxtacellular labeling with neurobiotin in eight neurons revealed multipolar cells 12-44 microm diameter with up to six primary dendrites. In three of eight neurons, a filled axon was present and coursed without branching toward the perimeter of the periaqueductal gray matter (PAG). The estrous cycle-related change in responsiveness to BIC and PG suggests that the panic circuitry in the PAG may become more responsive to panicogenic agents during estrus and late diestrus as a consequence of a decrease in the intrinsic level of inhibitory GABAergic tone. The findings may have implications for understanding the neural processes that underlie the development of premenstrual dysphorias in women.

[Mutation of the RET proto-oncogene in type 2A multiple endocrine neoplasia Chinese families and the application of pentagastrin stimulation test in diagnosis and follow-up].

OBJECTIVE: To detect the mutations of RET proto-oncogene in two Chinese families with multiple endocrine neoplasia type 2A (MEN2A) and to study the clinical application of pentagastrin stimulation test in the diagnosis and follow-up of MEN2A. METHODS: In this study, there were 4 patients clinically diagnosed as MEN2A in the two pedigrees. PCR and direct gene sequencing of PCR products with an automated DNA sequencer were used to scan the entire 21 exons of RET proto-oncogene in the leukocyte DNA of 6 members of the two families. Blood calcitonin was measured and pentagastrin stimulation test carried out in all the 4 clinically diagnosed patients. RESULTS: Two missense mutations were detected in exon 11 of the RET proto-oncogene. One was C634R in the 2 patients from one MEN2A family and the other was C634Y in 2 patients from another family. The basal serum calcitonin was very high (400.5 - 13 510.7 ng/L, normal range 16.6 - 132.8 ng/L) in the MEN2A patients and after pentagastrin stimulation the serum calcitonin level was significantly higher (494.1 - 33 901.9 ng/L). CONCLUSIONS: Two mutations (C634R and C634Y) were detected in the patients of two families with MEN2A. Pentagastrin stimulation test was helpful for the diagnosis and follow-up after operative treatment of MEN2A.

Cardiovascular and respiratory responses to a panicogenic agent in anaesthetised female Wistar rats at different stages of the oestrous cycle.

In urethane-anaesthetised female Wistar rats, intravenous injection of the panicogenic CCK(B) receptor agonist pentagastrin (0.002-80 microg/kg) evoked a dose-related increase in blood pressure, heart rate and ventilation. The response was blocked in the presence of the selective CCK(B) receptor antagonist CR2945 (1 mg/kg i.v.). The same pattern of cardiovascular and respiratory changes was evoked by microinjection of pentagastrin (0.3 nmol in 250 nL) into the dorsal half of the periaqueductal grey matter (PAG). The effect of intra-PAG administration of pentagastrin was also abolished following injection of CR2945 (1 mg/kg, i.v.). Responsiveness to systemically administered pentagastrin was enhanced in rats in late dioestrus. At the highest dose tested (80 microg/kg), the pressor response, tachycardia and tachypnoea evoked in rats in late dioestrus was significantly higher than rats in proestrus. For rats in oestrus, the pressor response and tachycardia but not tachypnoea were also significantly larger than the response evoked in rats in early dioestrus. The results suggest that the dorsal half of the PAG (dPAG) plays a key role in mediating the cardiovascular and respiratory responses evoked by systemically administered CCK(B) agonists. The enhanced responsiveness to panicogenic agents during late dioestrus may be related to changes in the functional responsiveness of gamma-aminobutyric acid (GABA)ergic circuitry in the dPAG due to plasticity of GABA(A) receptor subunit expression as a consequence of falling progesterone levels.

Pentagastrin-induced protein synthesis in the parotid gland of the anaesthetized rat, and its dependence on CCK-A and -B receptors and nitric oxide generation.

In parotid glands of pentobarbitone-anaesthetized rats, the incorporation of [3H]leucine into trichloroacetic acid-insoluble materials, reflecting protein synthesis, increased by 17% (compared to saline-treated rats) in response to infusion of pentagastrin (20 microg kg(-1), i.v. for 1 h) under muscarinic and alpha- and beta-adrenoceptor blockade. Both the CCK-A receptor antagonist lorglumide (48 mg kg(-1), i.v.) and the CCK-B receptor antagonist itriglumide (5.5 mg kg(-1), i.v.), given separately, prevented the expected increase in pentagastrin and, in addition, reduced the glandular protein synthesis by 16 and 12%, respectively, below the level of saline-treated rats. In rats treated with saline only, the glandular protein synthesis was reduced by 22% by the CCK-A receptor antagonist and by 17% by the CCK-B receptor antagonist; combined, the two antagonists caused no further reduction (20%). There was no increase in the glandular protein synthesis of pentagastrin-treated rats compared to that of the saline-treated rats when both groups of rats were exposed to a combination of the two types of CCK receptor antagonists. In pentagastrin-treated rats, the protein synthesis in the parotid glands was 23% less in the presence of the non-selective nitric oxide (NO) synthase inhibitor L-NAME (30 mg kg(-1), i.v.) than in its absence; the result was the same (23%) when the neuronal NO synthase inhibitor Nomega-propyl-L-arginine (N-PLA; 30 mg kg(-1), i.v.) replaced L-NAME. The protein synthesis in rats treated with saline only was not reduced by L-NAME; nor was the protein synthesis of saline-treated rats different from that of pentagastrin- and L-NAME-treated rats. Thus, under 'basal' conditions, a portion of the glandular protein synthesis, as well as the whole increase in synthesis in response to administration of pentagastrin, engaged both types of CCK receptors. Furthermore, NO generation, owing to neuronal NO synthase activity, was required for the increase to occur in response to pentagastrin, whereas a non-NO-dependent pathway was responsible for the protein synthesis under 'basal' conditions.