Ho Kalkman

Cloning and functional characterization of the rat stomach fundus serotonin receptor.

A DNA segment homologous to the third exons of the serotonin 1C and 2 receptor genes was isolated from a mouse genomic library. The positions of the introns flanking these exons were conserved in the three genes. To examine whether the new fragment was part of an active gene, we used a quantitative PCR protocol to analyse rat RNAs from different tissues and ages. The gene was expressed in stomach fundus at an abundance of 1 × 10(5) mRNA molecules. This tissue contracts in response to serotonin via a receptor that has previously resisted classification. We constructed a cDNA library from rat stomach fundus and isolated clones containing 2020 bp inserts with open reading frames of 465 amino acids comprising seven putative membrane‐spanning regions. The protein was transiently expressed in COS cells and binding of serotonergic ligands to the membranes was analysed. The pharmacological profile resembled that described for the serotonin‐stimulated contraction of the stomach fundus. After expression of this receptor in Xenopus oocytes, the application of serotonin triggered the typical chloride current which presumably results from the activation of phospholipase C. The coupling to this response system was less efficient than that of the 5‐HT1C or 5‐HT2 receptors.

ORGANIC AND INORGANIC CALCIUM-ANTAGONISTS REDUCE VASOCONSTRICTION INVIVO MEDIATED BY POSTSYNAPTIC ALPHA-2-ADRENOCEPTORS

SummaryThe influence of various calcium antagonists and divalent metal cations on the pressor responses induced by the selective α1-adrenoceptor agonist methoxamine and the selective α2-adrenoceptor stimulating agent B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine) was studied in pithed rats.1.The calcium antagonists verapamil, D 600 and nifedipine, when given intraarterially (i.a.) in doses up to 1 mg/kg did not influence the pressor effects of methoxamine. Only higher amounts of these calcium antagonistic drugs (1–3 mg/kg i.a.) somewhat reduced this pressor response.2.The vasoconstriction due to B-HT 920, as reflected by the increase in diastolic pressure, was markedly inhibited by verapamil, D 600 and nifedipine in a dose-dependent manner. In low doses a parallel displacement to the right was observed, whereas in higher amounts the shift was non-parallel.3.The divalent cations Mn2+, Ni2+ and Co2+ (0.05–0.15 mmol/kg i.a.) hardly affected the pressor effect of methoxamine, whereas B-HT 920-induced vasoconstriction was highly sensitive to these metal ions. La3+ and Mg2+ were ineffective.4.The calcium antagonists verapamil, D 600 and nifedipine displayed only minor affinities for [3H]prazosin (α1) as well as [3H]clonidine (α2) binding sites of rat brain membranes.5.It is concluded that an influx of extracellular Ca2+ is necessary for the vasoconstriction in vivo initiated by stimulation of vascular postsynaptic α2-adrenoceptors. On the other hand, vasopressor responses to α1-adrenoceptor stimulation are not directly dependent on a transmembrane influx of calcium ions.

Effect of converting enzyme inhibition and angiotensin receptor blockade on the vasoconstriction mediated by alpha 1-and alpha 2-adrenoceptor stimulation in pithed normotensive rats

SummaryThe effect of functional impairment of the renin-angiotensin system on the vasoconstriction mediated by postsynaptic α1 and α2-adrenoceptors in pithed normotensive rats was studied. Selective α1-adrenoceptor stimulation was induced by intravenously administered cirazoline, whereas B-HT 920 was used as a selective agonists at α2-adrenoceptors. The angiotensin converting enzyme was inhibited by intravenous treatment of the pithed rats with captopril, teprotide or enalapril. Blockade of angiotensin receptors was produced by intravenously applied [Sar1 Ala8]angiotensin II (saralasin). Pretreatment with angiotensin converting enzyme inhibitors or with saralasin in doses which produced a maximal reduction in basal diastolic blood pressure, only slightly attenuated the hypertensive response to cirazoline. In contrast, these drugs provoked a most significant reduction of the α2-adrenoceptor mediated vasoconstriction. Restoration of the basal diastolic blood pressure by intravenous infusion with angiotensin II or with vasopressin completely reversed the inhibitory effect of captopril on the vasopressor response to B-HT 920. One hour after bilateral nephrectomy, captopril still reduced the α2-adrenoceptor mediated vasoconstriction. However, 18–24 h after bilateral nephrectomy, captopril had no additional inhibitory effect on the vasopressor response to selective α2-adrenoceptor stimulation. It is concluded that in pithed normotensive rats the pressor response to α2-adrenoceptor stimulation is significantly potentiated by endogenous angiotensin II, even at low circulating levels of the octapeptide. The modulatory action of angiotensin II on the α-adrenoceptor mediated vasoconstriction probably represents an effect on the basal arteriolar muscular tone rather than a specific interaction.

DIFFERENTIAL EFFECT OF CALCIUM ENTRY BLOCKERS ON ALPHA-1-ADRENOCEPTOR-MEDIATED VASOCONSTRICTION INVIVO

SummaryThe effects of the calcium entry blockers nifedipine, (−)-verapamil and the dihydropyridine derivative PY 108-068 were evaluated on the increase in diastolic pressure of pithed normotensive rats caused by the selective α1-adrenoceptor agonists cirazoline, (−)-phenylephrine, (±)-erythro-methoxamine, (−)-amidephrine and St 587 [(2-chloro-5-trifluoromethylphenylimino)-2-imidazolidine] as well as by the mixed α1/α2-adrenoceptor agonists clonidine and DPI [(3,4-dihydroxyphenylimino)-2-imidazolidine]. The calcium entry inhibitors (up to 3 mg/kg) caused 3- to 5-fold, parallel rightward shifts of the log dose-pressor effect curves to cirazoline, (−)-phenylephrine, (±)-erythro-methoxamine and (−)-amidephrine accompanied by only a slight depression of the maximal pressor response. In contrast, the calcium entry inhibitors produced a dose-dependent profound depression of both maximum and slope of the log dose-pressor response curves to St 587 and clonidine. For DPI about 10-and 100-fold parallel displacements to the right without reduction of the maximum were found following treatment with 1 and 3 mg/kg of nifedipine, respectively. Infusion of vasopressin to counteract the vasodilatory action produced by the calcium entry inhibitors did not significantly change the pattern of interference observed under the conditions of decreased baseline diastolic pressure. The results indicate that α1-adrenoceptor-mediated vasoconstriction in the pithed normotensive rat, which is characterized by its sensitivity to blockade by prazosin and its relative insensitivity to antagonism by yohimbine or rauwolscine, can be subdivided into two distinct processes which are differentially influenced by blockade of calcium entry. It is suggested that cirazoline, (−)-phenylephrine, (±)-erythro-methoxamine and (−)-amidephrine initiate vasoconstriction predominantly by a mechanism independent of extracellular calcium influx. On the other hand, St 587 and clonidine increase arterial pressure via a mechanism which seems to be mainly governed by an entry of extracellular calcium ions. DPI probably triggers both processes of vasoconstriction in the intact circulatory system of the pithed normotensive rat.

CENTRAL 5-HT1A RECEPTORS AND THE MECHANISM OF THE CENTRAL HYPOTENSIVE EFFECT OF (+)8-OH-DPAT, DP-5-CT, R28935, AND URAPIDIL

This study investigated the central hypotensive effects of drugs that possess a high affinity for central 5-hydroxytryptamine (5-HT1A) binding sites; (+)8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), N,N-dipropylcarboxamidotryptamine (DP-5-CT), erythro-1-(1-[2(1,4-benzodioxan-2-yl)-2-hydroxyethyl]- 4-piperidyl)-2-benzimidazolinone (R28935) and urapidil proved to possess high affinity and selectivity for central 5-HT1A binding sites, labeled by [3H]8-OH-DPAT. (+)8-OH-DPAT (0.1-10 micrograms/kg) given through the left vertebral artery of chloralose-anesthetized cats, lowered blood pressure by a biphasic dose-response curve. When given systemically, 10- to 100-fold higher doses of (+)8-OH-DPAT were necessary to obtain the same hypotensive effect when compared with central administration. Besides 8-OH-DPAT, R28935, DP-5-CT and urapidil also lowered blood pressure by a central mechanism in doses that were ineffective when given systemically. The central hypotensive effect of 0.3 micrograms/kg (+)8-OH-DPAT, 3 micrograms/kg DP-5 CT, and 3 micrograms/kg R28935 could be blocked by 100 micrograms/kg (-)pindolol, indicating that central 5-HT1A receptors are involved. High doses of (+)8-OH-DPAT (3-10 micrograms/kg) can also lower blood pressure by activating central alpha 2-adrenoceptors. The hypotensive effect of 300 micrograms/kg urapidil given through the vertebral artery could not be blocked by (-)pindolol. These results indicate the involvement of central 5-HT1A receptors in the mechanism of the central hypotensive activity of (+)8-OH-DPAT, DP-5-CT, and R28935.

Captopril impairs the vascular smooth muscle contraction mediated by postsynaptic α2-adrenoceptors in the pithed rat

Since two different types of α-adrenoceptors (α1- and α2-) subserve vasoconstriction in pithed rats and noradrenaline behaves as a non-selective agonist of both receptors (for review see Timmerman and van Zwieten, 1981), we have investigated this interaction between noradrenaline and captopril in more detail to identify the class of α-adrenoceptor involved. This study confirms the findings reported recently (Antonaccio and Kerwin, 1981; Clough et al., 1981) that captopril inhibits postjunctional vasocontrictor responses to noradrenaline in pithed rats. However, captopril interfered with the vasoconstriction elicited by noradrenaline only in those animals in which the vascular postsynaptic α2-adrenoceptor system could be triggered (saline-and prazosin-treated rats). Captopril was without effect after removal of the contribution of post-synaptic α2-adrenoceptor stimulation to the in vivo vascular smooth muscle contraction due to (-)-noradrenaline (rauwolscine-treated rats). These results indicate that captopril selectively impairs the vasoconstriction subserved in vivo by postjunctinal α2-adrenoceptors. The vasoconstrictor process initiated by α1-adrenoceptor activation is not affected by captopril.

EFFECT OF CAPTOPRIL ON SYMPATHETIC NEUROTRANSMISSION IN PITHED NORMOTENSIVE RATS

Captopril significantly diminished the basal diastolic blood pressure and the vasopressor response to electrical stimulation of the thoracic-lumbar spinal cord in pithed normotensive rats. The reduction of the hypertensive response to electrical stimulation was more pronounced after bilateral adrenalectomy. Captopril also diminished the vasopressor response to intravenously administered (-)-noradrenaline. Pretreatment of the animals with indomethacin had no effect on the vasopressor response to electrical stimulation and did not affect the sympathoinhibition of captopril. After bilateral nephrectomy, 18-24 h previously, the basal diastolic blood pressure and the vasopressor response to electrical stimulation were reduced and captopril had no additional inhibitory effect on these parameters. In indomethacin-pretreated animals with intact kidneys, restoration of the basal diastolic blood pressure with angiotensin II (AII) completely abolished the sympathodepressive effect of captopril. When the reduction in basal diastolic blood pressure with captopril was prevented by vasopressin, converting enzyme inhibition had no depressive effect on the hypertensive response to intravenously administered (-)-noradrenaline and did not influence sympathetic neurotransmission in animals with intact adrenals. However, a small, but significant reduction of the hypertensive response to electrical stimulation by captopril was still detectable in bilaterally adrenalectomized rats. The results suggest that endogenous AII facilitates sympathetic neurotransmission in vascular smooth muscle of pithed normotensive rats. However, the modulatory action of endogenous AII largely results from an effect on basal arteriolar smooth muscle tone and should, therefore, be considered as non-specific facilitation. Genuine prejunctional facilitation of the sympathetic neurotransmission in vascular smooth muscle can only be observed after bilateral adrenalectomy but this effect of endogenous AII appears of minor significance, at least in pithed normotensive rats.

α1/α2-Adrenoceptor agonist selectivity of mono- and dihydroxy-2-N,N-di-n-propylaminotetralins

The pressor activities and the identity of the postjunctonal α-adrenoceptors involved were determined for a series of congeneric mono- and dihydroxy-substituted 2-N,N-di-n-propylaminotetralins and N,N-di-n-propyldopamine (DPDA) following i.v. administration to pithed normotensive rats. The affinity for α1- and α2-adrenoceptor-like binding sites was obtained from radioligand displacement studies. The 5- and 7-OH substituted tetralins as well as DPDA were reasonably potent and about equieffective pressor agents. The 6-OH congener had almost no vasoconstrictor effects whereas the 8-OH positional isomer occupied an intermediate position. The 5,6- and 6,7-di-OH analogs very effectively raised the diastolic pressure of pithed rats. On account of the inhibition exerted by prazosin (0.1 mg/kg) and yohimbine (1 mg/kg) the 5- and 7-OH isomers as well as DPDA can be classified as mixed α1/α2-adrenoceptor agonists, the α1-adrenoceptor-stimulating potency being more pronounced, especially for the 5-OH congener. In addition, a significant contribution of serotonin receptors to the pressor responses to the 8-OH compound was detected. Similarly, α2-adrenoceptors were mainly responsible for the vasoconstriction caused by the 6,7-di-OH isomer, whereas the 5,6-di-OH congener very selectively stimulated this α2-type receptor in the lower dose range and α1-adrenoceptor stimulation predominated at higher doses of this agonist. The 6,7-di-OH compound failed to activate vascular postjunctional β2-adrenoceptors. The results indicate that the α1/α2-adrenoceptor agonist activity depends on the position(s) and the number of hydroxy groups present as well as on the alkyl substitution at the amino function. 2-N,N-Di-n-propylamino-6,7-dihydroxytetralin may be a more suitable α2-adrenoceptor selective agonist than M-7.

Zur Pharmakologie vasodilatatorisch wirksamer Pharmaka; neuere Entwicklungen

Die durch Pharmaka bewirkte Erweiterung von Blutgefasen kann aus verschiedenartigen therapeutischen Grunden erwunscht oder erforderlich sein. Bei der Behandlung der arteriellen Hypertonie ist ein erneutes Interesse an Gefaserweiternden Substanzen offensichtlich und aus haemodynamischen Grunden durchaus gerechtfertigt. Auch bei der Therapie schwererer Formen der Myokardinsuffizienz haben Vasodilatotoren sich nebst Herzglykosiden und Saluretica einen gewissen Platz erworben, der auch theoretisch gut begrundet ist. Enttauschend sind allerdings die therapeutischen Ergebnisse bei der Anwendung von gefaserweiternden Pharmaka bei peripheren und zerebralen Durchblutungsstorungen, die auf einem atherosklerotischen Prozes beruhen. Dagegen kann eine nicht-atherosklerotische Durchblutungsstorung, wobei eine vasospastische Komponente im Spiel ist, wie zum Beispiel beim Syndrom von Raynaud, mitunter gunstig auf Vasodilatotoren reagieren.