Expression of somatostatin receptors on human pituitary adenomas in vivo and ex vivo.

UNLABELLED: The distribution and biologic activity of somatostatin receptor subtypes (SSTR) in pituitary adenomas is not clarified, especially regarding clinically non-functioning adenomas (NFPA). We therefore characterized SSTR in human pituitary adenomas by combining molecular biology and in vivo scintigraphy. Co-expression of gonadotropin-releasing hormone receptor (GnRH-R) mRNA was also assessed to see whether this feature was associated with adenoma subtype and SSTR status. Pituitary tumor biopsies were obtained during transsphenoidal adenomectomy from 21 patients (11 NFPA, 7 acromegalics, 2 prolactinomas, 1 Cushing's disease). Expression of mRNA encoding the 5 known SSTR subtypes and the GnRH-R was determined by RT-PCR. Twelve patients also underwent a pre-operative somatostatin receptor scintigraphy. Most adenomas (no.=18) expressed mRNA for more than one SSTR. SSTR2 mRNA was expressed in 18 cases, whereas SSTR4 was absent in all but one. SSTR3 was frequently expressed in NFPAs. Somatostatin receptor scintigraphy was positive in most cases, and with a significantly higher uptake index in GH-producing adenomas all of which expressed SSTR2 mRNA. The uptake index appeared to be related to receptor density rather than tumor volume. Expression of GnRH-R mRNA was found in both NFPAs and GH-producing adenomas and was not significantly associated with a particular SSTR subtype population. IN CONCLUSION: 1) the distribution of SSTR is not significantly different between NFPA and GH-producing adenomas; and 2) somatostatin receptor scintigraphy reveals a higher uptake in GH-producing adenomas which is not significantly related to either SSTR distribution or tumor volume.

Gene transcription of receptors for growth hormone-releasing peptide and somatostatin in human pituitary adenomas.

Growth hormone (GH)-releasing peptides (GHRP) or secretagogs (GHS) constitute a family of synthetic compounds with potent and specific GH releasing activity. The receptor (GHS-R) has recently been cloned even though the endogenous ligand remains to be identified. GHRPs act both at the hypothalamic and the pituitary level through mechanisms involving amplification of GH-releasing hormone activity and functional somatostatin antagonism. In the present study we examined the co-expression of messenger RNA (mRNA) for GHS-R and all 5 somatostatin receptor subtypes (sstr 1-5) in 28 human pituitary tumors by RT-PCR. GHS-R transcription was detected in 11 out of 12 somatotroph adenomas and in 2 out of 2 prolactinomas, whereas GHS-R expression was detected in only 2 out of 14 clinically nonfunctioning adenomas (NFPA), and no expression was seen in the only ACTH secreting adenoma. Almost all tumors expressed sstr 2 mRNA (n = 24), whereas only 1 tumor expressed sstr 4 mRNA. The expression of sstr 3 mRNA was inversely associated with GHS-R expression (P < 0.001), which could be attributed to a high prevalence of sstr 3 expression in NFPA. This study suggests that GHS-R expression is predominantly observed in somatotroph adenomas and much less so in NFPA. Moreover, the presence of a distinct pattern of somatostatin receptor subtype co-expression is suggested, which may provide a molecular basis for the complex interaction between GHRPs and somatostatin.

Involvement of K+ channels in the relaxant effect of vasoactive intestinal peptide and atrial natriuretic peptide in isolated guinea-pig trachea.

The possible contribution of K+ channel activation to airway smooth muscle relaxation induced by vasoactive intestinal peptide (VIP) and atrial natriuretic peptide (ANP) was investigated in isolated guinea-pig trachea. Concentration-relaxation (CR) curves were assessed in preparations precontracted by 30 mM K+, 124 mM K+ or histamine either alone or in the presence of a K+ channel blocker: iberiotoxin (IbTX), glipizide, tetraethylammonium (TEA) or Ba2+. VIP completely relaxed contractions induced by histamine but had a lower effectiveness against those induced by 30 mM K+ and 124 mM K+. IbTX and TEA shifted the CR curve for VIP 5 and 14 times to the right, respectively. Glipizide and Ba2+ did not significantly antagonize the action of VIP. ANP relaxed contractions induced by histamine and 30 mM K+ but failed to relax those elicited by 124 mM K+. IbTX and TEA shifted the CR curve for ANP 8 and 46 times to the right, respectively. Glipizide and Ba2+ suppressed the maximal effect produced by ANP, and glipizide also shifted the CR curve to the left. The K+ channel opener levcromakalim relaxed tracheal contractions induced by histamine and 30 mM K+ but not those induced by 124 mM K+. Glipizide caused a 5-fold rightward shift of the CR curve for levcromakalim whereas IbTX shifted the curve to the left and increased the maximal relaxant effect. The Ca2+ channel blocker isradipine completely relaxed contractions induced by 30 mM K+ and 124 mM K+ but only partially relaxed those contracted by histamine. All four K+ channel blockers increased the maximal relaxant effect and shifted the CR curve for isradipine to the left. The results suggest that airway smooth muscle relaxation produced by VIP and ANP involves activation of large-conductance Ca(2+)-activated K+ channels (BKCa) and further that ANP may possibly activate other types of K+ channels additional to BKCa.

Effects of levcromakalim and glibenclamide on paced guinea-pig atrial strips exposed to hypoxia.

Isolated strips of guinea-pig atrial myocardium were mounted in isometric myographs and electrically paced for measurements of myocardial contractile function. Levcromakalim, a K+ channel opener, completely inhibited the contractile force in a concentration-dependent way (EC50 = 15 microM). Glibenclamide (3 microM), a blocker of ATP-regulated K+ channels (KATP), caused a 5-fold rightward shift of the concentration-effect curve. Exposure of the atrial strips to hypoxia caused a time-dependent loss of contractility from 100% to a minimum level of 60% within 12 min. Levcromakalim (1 microM, 3 microM and 10 microM) concentration-dependently enhanced the hypoxia-induced inhibition of contractile function whereas levcromakalim (0.01 microM and 0.1 microM) had no significant effect. In the presence of levcromakalim (10 microM) hypoxia reduced the contractile force to 25%. Glibenclamide (3 microM) totally antagonized the enhancing effect of levcromakalim. When hypoxia was induced in glucose-free Krebs solution with 2-deoxyglucose, the myocardial contractility was completely suppressed within 12 min. Glibenclamide by itself (3 microM) failed to influence the myocardial response to hypoxia both in normal Krebs solution and under conditions of impaired glycolysis. The results indicate that levcromakalim by activation of myocardial ATP-regulated K+ channels accelerates and enhances the hypoxia-induced inhibition of myocardial contractile function. This effect may possibly contribute to the mechanism by which K+ channel openers exert cardioprotection. The results further suggest that mechanisms different from activation of KATP take a major part in the depressant mechanical response to hypoxia and glycolytic blockade in the guinea-pig atrial myocardium.

Inhibition by cromakalim, pinacidil, terbutaline, theophylline and verapamil of non-cholinergic nerve-mediated contractions of guinea-pig isolated bronchi.

Contractions induced by electrical field stimulation of sensory non-cholinergic excitatory nerves in guinea-pig isolated bronchi are due to the release of substance P (SP) and related tachykinins. Release of such neuropeptides are thought to play a pathophysiological role in asthma. Two K+ channel openers cromakalim (pD2 = 6.45; Emax = 95%) and pinacidil (pD2 = 6.06; Emax = 87%) were shown to concentration-dependently inhibit non-cholinergic nerve-mediated contractions in guinea-pig bronchi in vitro. Cromakalim (pD2 = 6.27; Emax = 25%) and pinacidil (pD2 = 6.03; Emax = 25%) each had a much lower inhibitory efficacy against contractions induced by exogenously applied SP but the same potency as found against contractile responses to non-cholinergic neurostimulation. Also the beta 2-adrenoceptor agonist terbutaline (pD2 = 8.29; Emax = 83%), the xanthine derivative theophylline (pD2 = 4.19; Emax = 100%) and the Ca2+ blocker verapamil (pD2 = 5.55; Emax = 100%) suppressed responses to non-cholinergic neurostimulation. Terbutaline (pD2 = 6.32; Emax = 74%), theophylline (pD2 = 3.25; Emax = 71%) and verapamil (pD2 = 4.01; Emax = 100%) had a 10-100-fold lower inhibitory potency against SP-induced contractions but each drug showed about the same efficacy as found against nerve-mediated contractions. Glibenclamide (1 microM) reversed the inhibitory effects of cromakalim and pinacidil on neurally-mediated contractions but did not influence the effects of terbutaline, theophylline and verapamil. The results demonstrate that cromakalim, pinacidil, terbutaline, theophylline and verapamil inhibit non-cholinergic excitatory neurotransmission in guinea-pig bronchi and suggest that they act preferentially at a pre-junctional site.

Glibenclamide inhibits hypoxic relaxation of isolated porcine coronary arteries under conditions of impaired glycolysis.

The possible involvement of ATP-sensitive K+ channels (KATP) in hypoxic relaxation of isolated porcine coronary arteries was investigated. Tubular segments taken from the left anterior descending artery were suspended in myographs for recording of isometric contractile force. Hypoxia (pO2 = 20.3 mm Hg +/- 0.5) produced a greater relaxation in preparations contracted by 30 mM K+ (49.7% +/- 7.2) compared with 124 mM K+ (19.9% +/- 2.2) which is compatible with the involvement of K+ channel activation in the mechanism of hypoxic relaxation. In a normal glucose-containing Krebs solution the KATP blocker glibenclamide (1 microM) failed to influence the hypoxic relaxation of preparations contracted by the thromboxane A2 analogue U-46619. Under conditions created to inhibit non-oxidative ATP production from glycolysis using a glucose-free Krebs solution containing 2-deoxyglucose (10 mM), the hypoxic relaxation was enhanced from 54.5% +/- 5.0 to 77.2% +/- 4.4. Under these conditions glibenclamide (1 microM) significantly inhibited the hypoxic relaxant response from 77.2% +/- 4.2 to 55.2% +/- 4.4 and prolonged the time until half-maximal relaxation from 5.5 min +/- 0.6 to 8.1 min +/- 0.6. A low concentration of the KATP opener levcromakalim (30 nM) failed to significantly potentiate the hypoxic relaxation. The adenosine receptor blocker theophylline (1 microM) or removal of the endothelium showed no effect on the hypoxic relaxation. In normal glucose-containing Krebs solution, indomethacin (10 microM) caused a small but significant inhibition of the hypoxic relaxation from 54.5% +/- 5.0 to 41.6% +/- 3.6.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic effects and pharmacokinetics of lemakalim in the isolated guinea-pig heart.

Myocardial effects of lemakalim were studied in the isolated retrogradely perfused and spontaneously beating guinea-pig heart. Pharmacokinetic analysis of drug accumulation and disposition showed two-compartment characteristics with phasic half-times of about 0.27 and 2.1 min., respectively. Myocardial clearance was 6.8 ml min.-1, and myocardial drug accumulation was 5-fold. Dynamic parameters were studied during increasing lemakalim concentrations from 1.4 nM up to 10 microM. Dynamic steady-states developed within 3-4 min. Increase of coronary flowrate showed an Emax of 185% with an EC50 of 60 nM. Lemakalim produced biphasic inhibitory actions on contraction velocity and amplitude. Emax and EC50 for the two phases were: 48% and 47% and 100 nM and 65 nM, respectively, for the first phase and 100% in both cases and 1.7 microM and 1.6 microM for the second phase. Heart frequency increased to a maximum of 15% above baseline at 0.3 microM. Oxygen consumption increased progressively at concentrations above 7 nM and reached a maximum of 175% at 0.3 microM. The frequency-corrected QT-interval was biphasically shortened to a maximal extent of 30% at the highest concentration. Myocardial efficiency expressed as the ratio of amplitude times frequency to oxygen consumption exhibited a progressive decline to about 20% of control values. PQ- and QRS-intervals showed only minor or no changes, respectively. No arrythmogenic effects were observed. The study demonstrated a rapid and very moderate accumulation of lemakalim in the guinea-pig heart accompanied by a marked increase in coronary flowrate and a progressive negative inotropic effect followed by reduced myocardial efficiency.

Isradipine dynamics and pharmacokinetics in the isolated rabbit heart.

The cardiac effects of increasing concentrations of isradipine (racemic) from 1.64 pM to 232 nM were studied in isolated spontaneously beating rabbit hearts. Inhibitory responses with regard to contraction amplitude, contraction velocity and oxygen consumption exhibited a biphasic progressive course at increasing drug exposure. Computer derived inhibitory Emax-values of the second phase were 104, 103 and 87% (IC50: 7.1, 6.3 and 28.7 nM), respectively, whereas those of the initial phase were 24.7, 25.9 and 19.5% (IC50: 0.012, 0.038 and 0.026 nM). A progressive inhibition of frequency reached a maximum of only 21%. The ECG-derived PQ-interval showed a rapid increase (maximum 46%) at drug concentrations above 1 nM. Complete AV-block and ventricular asystolia occurred in half of the hearts at the second highest (99 nM) and in all except one at the highest concentration. SA-node activity was retained in 9 of 10 hearts at the second highest and in 3 at the highest drug exposure. The QRS-and the frequency-corrected QT-interval did not increase significantly. Coronary flow-rate showed no initial increase, but a decrease to 70% of control at the highest concentration. Supplementary in vitro studies on rabbit coronary artery ring-preparations contracted with 124 mM K+ showed, however, an relaxant Emax-value for isradipine of about 100% and an inhibitory EC50-value of 0.63 nM with a 'Hill' coefficient of 1.1. At toxic concentrations isradipine showed a kinetic monophasic accumulation in the rabbit heart of about 44-fold with a half-time of 10.6 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of osmolarity of solutions used for K+ contraction on relaxant responses to pinacidil, verapamil, theophylline and terbutaline in isolated airway smooth muscle.

Concentration-relaxation profiles for pinacidil, verapamil, terbutaline and theophylline were studied in guinea-pig trachealis contracted by two commonly applied techniques for K+ depolarization. All drugs were much less effective on contractions induced by hyperosmolar 124 mMn K+ solution (added KCl) than on contractions elicited by an isoosmolar 124 mM K+ Krebs solution (substituted KCl). The maximal relaxant responses were (isoosmolar K+/hyperosmolar K+): pinacidil 100%/40%, verapamil 100%/60%, theophylline 100%/0%, terbutaline 50%/0%. Addition of mannitol to establish the same hyperosmolarity as with 124 mM KCl also produced contraction of guinea-pig trachealis. Concentration-relaxation curves for the drugs on mannitol-induced contractions had close resemblance to those obtained in hyperosmolar 124 mM K+ solution. When contraction was elicited by 30 mM K+, pinacidil showed seven times higher relaxant potency in hyperosmolar compared to isoosmolar solution, whereas the relaxant responses to verapamil, theophylline and terbutaline were not influenced by osmolarity. When K+ depolarization is used as a tool for evaluation of drug action in airway smooth muscle, the two different techniques produce dissimilar results. The influence of hyperosmolarity per se appears to be an important and unwanted feature when K+ depolarization is produced by addition of KCl.

Cardiac uptake kinetics and possible dynamic effects of a new cerebral antiischaemic compound FG 9202 (NBQX) studied in the isolated rabbit heart.

Accumulation of the putative drug FG 9202 in isolated rabbit hearts showed monophasic exponential kinetics with a half-life of only 0.59 min. The disposition showed a three-phasic exponential time course with half-lives of 0.34, 1.51 and 15.8 min, respectively, which was interpreted as three-compartment kinetics. FG 9202 accumulated only about 3 times in the myocardium at steady-state with 51, 32 and 17% referable to a superficial and two deeper myocardial drug pools. The drug did not significantly affect contraction amplitude or velocity of contraction at increasing concentrations up to 40.6 micrograms.ml-1 (119 microM). Heart beating frequency decreased slightly but only significantly at some of the higher concentrations. Neither dromotropic, bathmotropic nor ischaemic ECG-effects were observed. Coronary flow-rate and myocardial oxygen consumption decreased at the highest drug concentrations. Myocardial efficiency expressed as the ratio of contractile parameters to oxygen consumption showed a minor but insignificant increase at the highest drug-exposure levels. Our findings indicate that FG 9202 is not potentially toxic to the isolated, spontaneously beating rabbit heart in vitro.

Differential relaxant responses to pinacidil of smooth muscle preparations contracted by a high concentration of potassium in isoosmolar and hyperosmolar solutions.

Contractions were produced in guinea-pig trachealis, aorta and pulmonary artery by depolarization with 124 mM K+ using two commonly applied techniques. Addition of KCl to the organ bath solution making it hyperosmolar induced slowly developing contractions, which were only weakly inhibited by pinacidil. Hyperosmolar mannitol-induced contractions showed similar characteristics. In contrast, contractions elicited by isoosmolar K+ Krebs solution developed more rapidly and could be completely suppressed by pinacidil (10(-6)-10(-3) M) in a concentration-dependent manner. The findings explain previously published discrepant results on the relaxant response to pinacidil of smooth muscle preparations contracted by high concentrations of K+, and indicate other mechanisms of action for pinacidil in addition to K+ channel opening, in the concentration range 10(-6)-10(-3) M.

Lack of effect of pinacidil on theophylline pharmacokinetics and metabolism in man.

Pinacidil, a pyridyl cyanoguanidine derivative, is a new antihypertensive vasodilator drug. It shares structural similarities with the histamine H2-receptor blocker cimetidine, an imidazole cyanoguanidine derivative, which is a potent inhibitor of cytochrome P-450 and of theophylline metabolism. In the present study the pharmacokinetics and metabolism of theophylline were determined in six healthy volunteers before and on the last day of oral pinacidil administration for two weeks. The dosage of pinacidil was 12.5 mg twice a day in the first week and 25 mg in the second. There were no significant changes in theophylline plasma clearance, terminal half-life or volume of distribution during pinacidil administration. Also the renal and metabolic clearance of theophylline and the formation clearances of the major theophylline metabolites in the urine (DMU, 1MU, 3MX) did not change significantly during administration of therapeutic doses of pinacidil.

Lack of effect of the vasodilator pinacidil on insulin secretion in healthy humans.

Pinacidil is a new antihypertensive vasodilator drug which is supposed to act by opening of ATP-sensitive and glibenclamide-sensitive K+ channels in vascular smooth muscle cell membranes. Similar K+ channels play an important role in insulin secretion from pancreatic islets cells. Inhibition of insulin secretion has been demonstrated with high concentrations of pinacidil in vitro. In the present study the insulin response to oral glucose were studied in six healthy subjects before and on the last day of 2 weeks treatment with pinacidil. The drug was given by the oral route 12.5 mg bid in the first week and 25 mg bid in the second. There were no significant changes in fasting blood levels of insulin or glucose, glucose-stimulated insulin secretion, or oral glucose tolerance during pinacidil administration. These results may suggest that pinacidil at therapeutic concentrations does not activate insulin regulating K+ channels in pancreatic islet cells.

Myocardial accumulation kinetics and pharmacodynamics in the isolated rabbit heart of a new inhibitor of dopamine reuptake, GBR 12909.

Myocardial accumulation of GBR 12909 showed monophasic exponential kinetics with a half-life of 93 min. The disposition followed a three-phasic exponential time-course with half-lives of 1.1, 17 and 98 min., respectively, which was interpreted as three-compartment kinetics. The drug accumulated 430 times in the myocardium at steady-state with 8, 30 and 61% of the drug amount referable to a central, superficial and two deeper myocardial drug pools. GBR 12909 produced concentration dependent (range 0.01 to 12400 nM) biphasic negative inotropic and chronotropic effects. The inhibitory Em-values with regard to contraction velocity were 42 and 105% with corresponding EC50-values of 29 and 688 nM and the related Hill-exponents were 0.6 and 1.1, respectively. Frequency and contraction amplitude related inhibitory Em-values were of similar size. Apparent dynamic steady states developed within about 17 min. Very marked monophasic negative dromotropic effects were observed with computer-derived inhibitory Em-values related to the electrocardiographic PQ- and QRS-intervals exceeding 100%. The frequency-corrected QTc-interval showed an initial increase of 10% but decreased to about 20% below control level at the highest two drug concentrations. Coronary flow-rate increased about 30% and then gradually decreased to near the control value. Oxygen consumption only decreased at the three highest concentrations. Our findings seem compatible with the view that GBR 12909 may possibly act in the myocardium as a membrane-stabilizer which causes inhibition of Na(+)- and Ca+(+)-influx over sarcolemma. Intracellular inhibition of Ca+(+)-liberation from organelles and other calcium depots also seems possible.

A comparison of the relaxant effects of pinacidil in guinea-pig trachea, aorta and pulmonary artery.

The relaxant activity of pinacidil, a proposed K+ channel opener, was compared in isolated guinea-pig trachea, aorta and pulmonary artery. In preparations precontracted by histamine or PGF2 alpha, pinacidil produced complete tracheal relaxation but only partial relaxation of vascular tissues. The order of responsiveness was: pulmonary artery greater than trachea greater than aorta. The slope of the pinacidil concentration-effect (C/E) curve was much steeper in the tracheal than in the vascular preparations. The pinacidil C/E curves for relaxation were similar when the three types of preparations were precontracted by 124 mM K+. Pretreatment with pinacidil caused a parallel shift of the tracheal histamine C/E curve to the right, whereas the maximal response to histamine was markedly depressed in the pulmonary artery.

Pinacidil uptake and effects in the isolated rabbit heart.

The myocardial accumulation of pinacidil showed one-compartment characteristics with a half-time of 1.11 min., whereas the disposition followed three-compartment kinetics with half-times for the relevant two redistributory and the terminal phases of 0.39, 1.51 and 5.44 min., respectively. At a steady-state drug concentration in the perfusate of 6.12 nmol ml-1, the average concentration of pinacidil in the myocardium was 20.6 nmol g-1. The accumulated amount could predictically be referred with 57% to a central and 31 and 12% to two peripheral (deeper) drug pools. The pharmacodynamic effects of pinacidil in the isolated perfused rabbit heart were studied at stepwise increasing concentrations from 0.15 to 100 microM. Coronary flowrate increased initially up to 24.5% at 1.5 microM pinacidil and then gradually decreased. Amplitude and velocity of contraction were both inhibited in a biphasic way up to 92.7 and 94.1%, respectively. Apparent dynamic steady states developed within 13-15 min. The computer-derived inhibitory Em-values related to the first phase were 49.2 and 52.4% and those related to the second phase were 111.7 and 108.3%, respectively. Heart frequency decreased monophasically and exhibited an inhibitory Em-value of 19.6%. Oxygen consumption decreased at pinacidil concentrations higher than 15 microM and the Em-value was 69.7%. The frequency-corrected QT-interval decreased biphasically and the related inhibitory Em-values were 8.6 and 58.7%. The QRS-interval did not change and the PQ-interval only showed a minor increase at the highest pinacidil concentration. Our findings are compatible with the concept of pinacidil being a potassium channel opener.

Effects of pinacidil on guinea-pig airway smooth muscle contracted by asthma mediators.

Pinacidil is a new antihypertensive, direct vasodilator drug which has been classified as a K+ channel opener. The present study demonstrated a concentration-dependent relaxant activity of pinacidil in guinea-pig tracheal preparations. The potency and efficacy of pinacidil depended on the agent used to induce tracheal tone. Tracheal preparations with spontaneous tone or precontracted by different asthma mediators were completely relaxed by pinacidil. A high potency was found in spontaneously contracted preparations (EC50 = 7.8 x 10(-7) M). The EC50 values ranged from 2.3 to 5.4 x 10(-6) M in histamine-, PGF2 alpha- or LTC4-contracted preparations. When tone was induced by carbachol, the EC50 was 2.1 x 10(-5) M. In contrast, pinacidil produced incomplete relaxation and had a low potency in preparations contracted by 30 or 124 mM K+ Krebs solutions. This effect profile differed from that seen with beta 2-receptor agonists, xanthines and Ca2+ antagonists in guinea-pig trachealis and seems compatible with K+ channel opening as a primary mode of relaxation for pinacidil in airway smooth muscle.