Altered contractile sensitivity of isolated bronchial artery to phenylephrine in ovalbumin-sensitized rabbits.

We tested the hypothesis that atopy and/or allergic lung inflammation enhances alpha1-adrenoceptor-mediated contractions of the bronchial artery. Bronchial arterial resistance vessels were isolated from rabbits that had undergone either systemic ovalbumin (OVA) sensitization followed by saline aerosol challenge (OVA/saline rabbits), or OVA sensitization followed by OVA aerosol challenge (OVA/OVA rabbits), or no sensitization followed by saline aerosol challenge (control rabbits). In OVA/OVA rabbits, bronchoalveolar lavage and lung histology revealed lymphocytic and eosinophilic inflammation. Arterial rings were contracted with phenylephrine (PE). In endothelium-intact arteries isolated from OVA/saline and OVA/OVA rabbits, PE responsiveness was enhanced compared with that of arteries isolated from controls. The nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester increased the contractile response to PE in all three experimental groups to a similar degree, suggesting that depressed NOS activity was not involved in the enhanced PE responsiveness in OVA/saline and OVA/OVA rabbits. After endothelium removal, arteries from OVA/saline and control rabbits showed similar PE responsiveness, indicating that the enhancement of PE responsiveness was endothelium dependent, possibly due to an endothelial constricting factor. In OVA/OVA rabbits, endothelium-denuded arteries showed decreased PE responsiveness compared with the other two groups; this difference was abolished by NG-nitro-L-arginine methyl ester. We conclude that systemic sensitization with OVA per se enhances PE-induced contractions of isolated bronchial arteries in rabbits by an endothelium-dependent mechanism and that allergic lung inflammation attenuates this effect by increased nonendothelial NOS activity.

Norepinephrine-induced contraction of isolated rabbit bronchial artery: role of alpha 1- and alpha 2-adrenoceptor activation.

The contractile effect of norepinephrine (NE) on isolated rabbit bronchial artery rings (150-300 microns in diameter) and the role of alpha 1- and alpha 2-adrenoceptors (AR) on smooth muscle and endothelium were studied. In intact arteries, NE increased tension in a dose-dependent manner, and the sensitivity for NE was further increased in the absence of endothelium. In intact but not in endothelium-denuded arteries, the response to NE was increased in the presence of both indomethacin (Indo; cyclooxygenase inhibitor) and NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], indicating that two endothelium-derived factors, NO and a prostanoid, modulate the NE-induced contraction. The alpha 1-AR antagonist prazosin shifted the NE dose-response curve to the right, and phenylephrine (alpha 1-AR agonist) induced a dose-dependent contraction that was potentiated by L-NAME or removal of the endothelium. The sensitivity to NE was increased slightly by the alpha 2-AR antagonists yohimbine and idazoxan, and this effect was abolished by Indo or removal of the endothelium. Similarly, contractions induced by UK-14304 (alpha 2-AR agonist) were potentiated by Indo or removal of the endothelium. These results suggest that NE-induced contraction is mediated through activation of alpha 1- and alpha 2-ARs on both smooth muscle and endothelium. Activation of the alpha 1- and alpha 2-ARs on the smooth muscle causes contraction, whereas activation of the endothelial alpha 1- and alpha 2-ARs induces relaxation through release of NO (alpha 1-ARs) and a prostanoid (alpha 2-ARs).

Glaucoma, capillaries and pericytes. 4. Beta-adrenergic activation of cultured retinal pericytes.

To characterize the relaxation of pericytes induced by beta-adrenergic stimulation, changes in the contractile tone of pericytes were quantified as a change in the wrinkling of an elastic silicone surface on which they were cultured. Isoproterenol produced relaxation of pericytes in a dose-dependent manner over a range of 5 nM to 1 microM. Low concentrations of the nonselective beta-blockers propanolol and timolol blocked the relaxation produced by isoproterenol. The specific beta 2-adrenergic component of isoproterenol-induced relaxation was shown by blockage with bromoacetyl alprenolol menthane. In contrast, atenolol and betaxolol, as relatively selective beta 1-adrenergic blockers, had no effect on the isoproterenol-induced relaxation.

Relaxation of retinal pericyte contractile tone through the nitric oxide-cyclic guanosine monophosphate pathway.

PURPOSE: Pericytes are contractile cells outside the endothelial lining of capillaries. The study investigated whether nitric oxide, known to cause relaxation of vascular smooth muscle cells, also relaxes pericytes. METHODS: Cultured bovine pericytes were exposed to a nitric oxide donor, sodium nitroprusside, and to 8-bromoguanosine 3':5'-cyclic monophosphate (8-bromo cGMP) in the presence and in the absence of methylene blue, an inhibitor of guanylate cyclase. Relaxation was assessed by counting the loss of wrinkles induced by pericytes when they are cultured on thin sheets of silicone. Intracellular cyclic guanosine monophosphate (cGMP) was measured by radioimmunoassay. RESULTS: Pericytes relaxed to sodium nitroprusside (3 x 10(-8) to 10(-4) M) in a concentration-dependent manner (ED50: 10(-6) M). The maximal response was characterized by the loss of almost all the wrinkles. The half-maximal relaxation was strongly inhibited by methylene blue (3 x 10(-7) M). Sodium nitroprusside (3 x 10(-6) M) caused a 14-fold increase in the intracellular concentration of cGMP, an effect inhibited by methylene blue (3 x 10(-7) M). The cGMP analogue, 8-bromo-cGMP (10(-5) and 3 x 10(-5) M) strongly relaxed the cells in a concentration-dependent manner, but the relaxation was not reduced by methylene blue (3 x 10(-7) M). CONCLUSIONS: Sodium nitroprusside, which releases nitric oxide, strongly relaxes pericytes through an increase of cGMP. Therefore, the presence of the endothelium-derived relaxing factor nitric oxide in the microcirculation can modulate the tone of pericytes, and it thus has the potential to influence blood flow in capillaries.

Role of endothelium and hyperpolarization in CGRP-induced vasodilation of rabbit ophthalmic artery.

The electromechanical effects of calcitonin gene-related peptide (CGRP) on intact and endothelium-denuded rabbit ophthalmic arteries were studied. CGRP inhibited norepinephrine (NE)-induced contractions. In intact arteries after washout of CGRP the contractile sensitivity to NE was increased. Conversely, in endothelium-denuded arteries, the relaxation induced by CGRP was prolonged, and after washout of CGRP the contractile sensitivity to NE was diminished. In intact arteries NE contractions were enhanced by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of endothelium-derived relaxing factor (EDRF) synthesis, and in the presence of L-NMMA, CGRP-induced relaxations resembled those seen in endothelium-denuded arteries. This result suggests that there is an increased EDRF synthesis in intact arteries during NE stimulation and that CGRP may inhibit either the synthesis or the activity of EDRF. High concentrations of CGRP hyperpolarized the smooth muscle membrane both in intact and endothelium-denuded arteries. Hyperpolarizations were blocked by glibenclamide, indicating that they are mediated by activation of ATP-sensitive K+ channels. However, glibenclamide had little effect on the CGRP-induced relaxation. These results suggest that in normal physiological conditions CGRP-induced relaxation of the rabbit ophthalmic artery is mediated mainly by mechanisms other than hyperpolarization.

Inhibitory effect of methysergide on calcitonin gene-related peptide-induced vasodilatation and ocular irritative changes in the rabbit.

1. Calcitonin gene-related peptide (CGRP) is involved in ocular neurogenic inflammation in the rabbit, causing vasodilatation in the anterior uvea, breakdown of the blood-aqueous barrier, increase in the intraocular pressure (IOP) and rise in the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content in the aqueous humour. So far there is no means of preventing these CGRP-induced ocular effects. 2. In the present study, the effect of intravenous methysergide (1-10 mg kg-1, b.w.) on CGRP-induced changes in the IOP, blood-aqueous barrier and cyclic AMP content in the aqueous humour was studied in vivo. The effect of methysergide on CGRP-induced vasodilatation both in vivo and in vitro was also investigated. 3. Methysergide decreased intraocular pressure but had only a transient effect on blood pressure. Methysergide decreased the regional blood flow in ocular tissues by 53-65%, but did not have such a vasoconstrictor effect in most extra-ocular tissues studied. 4. Methysergide inhibited CGRP-induced vasodilatation, increase in the IOP, breakdown of the blood-aqueous barrier and increase in the cyclic AMP content in the aqueous humour in vivo. 5. In vitro, methysergide alone did not have effects on the vascular tone in isolated ophthalmic artery of rabbit. However, it potentiated noradrenaline (NA)-induced contraction. There were no differences in the IC50 values for CGRP on the NA-induced contraction in the presence and absence of methysergide, indicating that methysergide has no direct effect on the vasorelaxant effect of CGRP in vitro. 6. The present study demonstrates that in the rabbit eye methysergide inhibits CGRP-induced changes.One inhibitory mechanism of methysergide may be to enhance the effect of a vasoconstrictor (NA) to antagonize the vasodilator effect of CGRP. The present findings suggest that a methysergide-sensitive mechanism may be used to limit some pathophysiological conditions in the eye that involve neurogenic inflammation and the release of CGRP.

Serotonergic responses in rabbit ophthalmic artery: a pharmacological characterization.

The contractile effect of serotonin (5-HT) on rabbit ophthalmic artery was studied. In a solution containing 20 mM K+ 5-HT induced a biphasic dose-response curve (DRC) in intact arteries. At normal extracellular K+ concentration ([K+]o), only high concentrations of 5-HT (greater than or equal to 1 microM) were able to induce contraction. In endothelium-denuded arteries 5-HT induced a DRC at normal [K+]o, which resembled that in intact arteries at 20 mM [K+]o. The high-potency portion of the 5-HT DRC was inhibited only by metitepine, whereas the low-potency portion was blocked by metitepine, methysergide, spiperone, and ketanserin, indicating that in this preparation the 5-HT1 receptor subtypes are more sensitive to 5-HT than the 5-HT2 receptor subtypes. Cyanopindolol had no effect on 5-HT-induced contraction. 8-Hydroxy-2-(di-n-propylamino)tetralin gave a contraction at high concentrations (greater than or equal to 0.1 microM), which was not blocked by cyanopindolol. The contractile response to 5-methoxytryptamine was similar to that for 5-HT. The results indicate that the 5-HT1 receptors of the ophthalmic artery belong either to 1C or 1D subtypes. 2-Methylserotonin, an agonist for 5-HT3 receptor, had no contractile effect on rabbit ophthalmic artery. The effect of prior exposure to 5-HT on norepinephrine (NE)-induced contraction was also studied. In intact arteries prior exposure to a low 5-HT concentration (10 nM) induced attenuation and prior exposure to a high 5-HT concentration (1 microM) gave potentiation of the NE-induced contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of leukotriene C4 on electromechanical activity and Ca2+ uptake in taenia coli.

The actions of leukotriene C4 (LTC4) on electromechanical activity and 45Ca2+ uptake in guinea pig taenia coli were investigated. The contractile action of LTC4 was abolished by the removal of extracellular Ca2+. LTC4 concentrations eliciting a maximal contraction in normal medium produced no response in preparations depolarized with KCl. In single sucrose gap studies, LTC4 increased both the frequency of electrical spiking and tension. These effects were blocked by the dihydropyridine Ca2+-channel antagonist PY 108-068 and by the leukotriene receptor antagonist FPL 55712. In double sucrose gap experiments, LTC4 caused a small depolarization without measurable change in membrane conductivity; increased spontaneous electrical activity was again accompanied by an increase in tension. LTC4 caused a detectable increase in 45Ca2+ uptake only at extracellular Ca2+ concentrations less than 1 mM, and this was again inhibited by PY 108-068 or FPL 55712. It is concluded that the contractile effects of LTC4 in guinea pig taenia coli occur as a consequence of its ability to open voltage-sensitive Ca2+ channels, an effect that may occur independently of membrane depolarization.

Calcium channels in thrombin-activated human platelet membrane.

Platelet-activating factor, 5-hydroxytryptamine, thromboxane A2, adenosine diphosphate and thrombin are known to activate platelets by stimulating calcium entry, but the nature of the entry pathways is unknown. We present the identification of single divalent cation channels from thrombin-activated human platelets. Membrane vesicles from unstimulated and thrombin-stimulated human platelets were incorporated in planar bilayers and unitary currents through single channels were measured. Divalent cation selective channels could only be demonstrated in thrombin-stimulated preparations. These channels share a number of properties in common with voltage-dependent calcium channels--a high degree of selectivity for divalent cations, a single channel conductance of about 10 pS (in 150 mM Ba2+) and sensitivity to blockade by inorganic calcium channel blockers such as Ni2+. In other respects, these channels are different as they are not voltage-dependent and are not blocked by 1,4-dihydropyridine calcium channel antagonists.

Vasopressor peptides and depolarization stimulated Ca2+-entry into cultured vascular smooth muscle.

45Ca-uptake was measured in monolayers of cultured rat aortic smooth muscle cells. Sufficient extracellular 45Ca could be removed by a 90 second cold La3+ was to reveal stimulation of 45Ca-uptake by high K+-depolarization and the vasopressor peptides angiotensin II and vasopressin. The high K+-stimulated 45Ca-influx was blocked by a dihydropyridine-type Ca2+-antagonist while that stimulated by angiotensin II or vasopressin was not. The 45Ca-influx stimulated by high K+-depolarization was additive to that stimulated by angiotensin II. Vasopressin and angiotensin II stimulated 45Ca-fluxes were not additive. It is concluded that vasopressor peptides stimulate Ca2+-entry through receptor operated Ca2+-channels which are distinct from voltage gated Ca2+-channels.

Vascular smooth muscle calcium channels.

Vascular smooth muscle Ca channels function in excitation-contraction coupling. A survey of recent literature reveals several types of excitable Ca channels. There are at least two plasmalemmal Ca channels that are primarily activated by depolarization. In addition, there also exists evidence for the presence of Ca channels in conduit arteries that are primarily activated by agonists. Under circumstances of compromised sarcoplasmic reticulum (SR) Ca accumulation, Ca that enters through the nonregulated Ca leak also contributes to tension development. The Ca release from the SR appears to be mediated by large Ca channels that are activated by free Ca, inositol-1,4,5-trisphosphate, and free ATP. The differential sensitivity to procaine suggests the presence of two separate excitable Ca channels in vascular smooth muscle SR in addition to a basal Ca leak. This presentation concludes with a theoretical model describing how vascular smooth muscle Ca metabolism may be altered in hypertension and how a Ca antagonist may lead to reduction of blood pressure.

Abnormal cellular calcium regulation in essential hypertension.

The plasticity of cellular Ca2+ control and the events regulated by [Ca2+]i and other messengers make it difficult to assign causative or consequential roles to deranged platelet Ca2+-linked processes in the pathophysiology of essential hypertension. Our studies in human platelets support an underlying membrane pathology as being causative since observed derangements including partial membrane depolarization and enhanced calcium influx, enhanced hormone responsiveness and coupling to adenylate cyclase, increased phosphoinositide metabolism, as well as stimulated Ca2+-ATPase extrusion activity are membrane associated systems. Modification of phosphoinositide metabolism may be a key factor accounting for the multifaceted membrane abnormalities and eventually contribute to the elevated cytosolic [Ca2+]i concentration in essential hypertension. Whether these membrane abnormalities can also be found in human smooth muscle cells has yet to be determined.

Biphasic Ca2+ response of adenylate cyclase. The role of calmodulin in its activation by Ca2+ ions.

The Ca2+-dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1-1.0 microM). At low Ca2+ (0.08-0.3 microM) adenylate cyclase was stimulated (Ka = 0.10 microM), whereas at higher Ca2+ (greater than 0.3 microM) the enzyme was inhibited to 70-80% control (Ki = 0.8 microM). Membrane fractions, prepared by washing in the presence of LaCl3 to remove endogenous calmodulin (approximately equal to 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 microM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin (Ka = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 microM). Prostaglandin E1 (PGE1) did not alter Ki or Ka values for Ca2+. Calmodulin did not alter the EC50 for PGE1 stimulation of adenylate cyclase but increased the Vmax (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3+-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.

Morphological changes produced in rat testis by anticancer drugs.

Testicular side effects of procarbazine (Proc: 50 or 200 mg/kg i.p.), vincristine (Vin: 0.15 or 0.6 mg/kg i.p.) or busulfan (Bu: 10 mg/kg p.o.) were examined by morphological methods 3 days, 1 week and weekly thereafter until week 10 after a single exposure. With Proc a degeneration of the germ cells, particularly of mid primary spermatocytes, was seen first. Morphogenesis of early spermatids was disturbed, especially of those subcellular elements depending on an intact RNA metabolism. Later, giant cells were frequent. Vin led first to a malformation of late spermatids and arrest of cell division of spermatocytes and especially of spermatogonia, indicating microtubule dysfunction. After 2 and 4 weeks Bu showed a disappearance of spermatogonia and early spermatocytes leading to a depletion of the germinal epithelium by maturation. Late effects were rather similar in all the groups.

Drug-induced histological changes in rat seminiferous tubular epithelium.

It is proposed to divide antispermatogenic compounds into four main groups on the basis of the morphological changes which they cause: a) Radiomimetic substances (e.g., Busulfan). These have a direct effect on spermatogonia and hence also on later sperm development. b) Substances inhibiting the meiotic and postmeiotic phases of spermatogenesis. These cause the seminal epithelium to disappear rapidly except for spermatogonia and Sertoli cells, which apparently remain intact (e.g., 20-438, an indenopyridine derivative). c) Substances acting hormonally (via pituitary). These severely damage Leydig cells, and there is a particularly pronounced degeneration of later stage spermatids. Sertoli cells are also affected. The tunica propria becomes thicker. The weights of the accessory sex glands decrease, and the pituitary weight increases. The peritubular tissue in the epididymis becomes thicker (e.g., 17 beta-estradiol). d) Vasoactive substances (e.g., cadmium chloride). These cause spot-like necrosis. Whole tubuli are destroyed (similar to infarction) whereas others can remain intact. Leydig cells also degenerate, and there is fibroblastic proliferation in the interstitium. The peritubular tissue in the epididymis becomes thicker and the secretion of clear cells is affected. Accessory glands lose weight.