Charlotte Fetscher

M3 muscarinic receptors mediate contraction of human urinary bladder

Since muscarinic receptors appear to be the physiologically most important control system for urinary bladder contraction, we have characterized the receptor subtype mediating contraction in response to the muscarinic agonist carbachol in the human bladder. Experiments were based on four antagonists, the non‐selective atropine, the M1‐selective pirenzepine, the M2‐selective methoctramine and the M3‐selective darifenacin. All antagonists yielded Schild‐plots with a slope close to unity. The order of potency (atropinedarifenacin>pirenzepine>methoctramine) as well as the estimated antagonist affinities suggested that contraction of the human bladder occurs predominantly if not exclusively via the M3 receptor.

Sphingosine-1-phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro

Sphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G‐protein‐coupled receptors, some of which were cloned and designated as Edg‐receptors. Sphingolipid‐induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid‐induced elevations in intracellular Ca2+ concentration were measured in cultured rat aortic smooth muscle cells. SPPC and SPP (0.1–100 μmol l−1) caused concentration‐dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC50 5.63±0.17 and Emax 49±3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC50 D‐erythro‐SPPC 5.69±0.08, L‐threo‐SPPC 5.31±0.06) and inhibited by pretreatment with pertussis toxin (Emax from 44±5 to 19±4%), by chelation of extracellular Ca2+ with EGTA and by nitrendipine (Emax from 40±6 to 6±1 and 29±6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (Emax from 87±3 to 70±4%). SPP and SPPC caused transient increases in intracellular Ca2+ concentrations in rat aortic smooth muscle cells in a pertussis toxin‐sensitive manner. Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca2+ concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin‐sensitive G‐proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

EFFECTS OF NORADRENALINE AND NEUROPEPTIDE Y ON RAT MESENTERIC MICROVESSEL CONTRACTION

We have studied the contractile effects of the sympathetic transmitter noradrenaline and its cotransmitter neuropeptide Y (NPY) given alone and in combination on isolated rat mesenteric resistance vessels (200–300 μm diameter). Noradrenaline and NPY each concentration-dependently contracted rat mesenteric microvessels (EC50 ≈ 800 nM and 10 nM, respectively), but noradrenaline caused considerably greater maximal effects than NPY (14.3 mN vs. 3.5mN). A low antagonistic potency of yohimbine indicated that the response to noradrenaline did not involve α2-adrenoceptors, and the subtype-selective antagonists 5-methylurapidil, tamsulosin and chloroethylclonidine indicated mediation via an α1A-adrenoceptor. Shallow Schild regressions for prazosin and 5-methylurapidil indicated that an α1-adrenoceptor subtype with relatively low prazosin affinity might additionally be involved. Studies with the NPY analogues PYY, [Leu31, Pro34]NPY and NPY18–36 demonstrated that NPY acted via a Y1 NPY receptor. In addition to its direct vasoconstricting effects NPY also lowered the noradrenaline EC50 but did not appreciably affect maximal noradrenaline responses indicating possible potentiation. The potentiating NPY response occured with similar agonist potency as the direct contractile NPY effects and also via a Y1 NPY receptor. The Ca2+ entry blocker nitrendipine (300 nM) reduced direct contractile responses to noradrenaline and NPY but did not affect the potentiation response to NPY.

Extracts from Rhois aromatica and Solidaginis virgaurea inhibit rat and human bladder contraction

Since extracts from the plants Rhois aromatica and Solidaginis virgaurea are being used in the phytotherapy of bladder dysfunction including the overactive bladder syndrome, and since muscarinic receptors are the main pharmacological target in the treatment of bladder dysfunction, we have investigated whether these extracts can inhibit carbachol-induced, muscarinic receptor-mediated contraction of rat and human bladder. In vitro contraction experiments were performed with rat and human bladder strips. Radioligand binding and inositol phosphate accumulation studies were done with cells transfected with human M2 or M3 muscarinic receptors. Both extracts concentration-dependently (final concentrations 0.01–0.1%) inhibited carbachol-induced contraction of rat and human bladder with insurmountable antagonism. Radioligand binding experiments and inositol phosphate accumulation studies with cloned receptors demonstrated direct but non-competitive effects on muscarinic receptors. Reductions of KCl-induced bladder contraction demonstrated that inhibition by the higher extract concentrations also involved receptor-independent effects. We conclude that extracts from Rhois aromatica and Solidaginis virgaurea inhibit muscarinic receptor-mediated contraction of rat and human bladder. While this could contribute to the beneficial effects of these extracts in patients with bladder dysfunction, such therapeutic effects remain to be demonstrated in controlled clinical studies.

Transient relaxation of rat mesenteric microvessels by ceramides

We have investigated the vasodilating effects of D‐erythro‐C2‐ceramide (C2‐ceramide) in methoxamine‐contracted rat mesenteric microvessels. C2‐ceramide (10–100 μM) caused a concentration‐dependent, slowly developing relaxation which reached maximum values after ∼10 min and partially abated thereafter. Endothelium removal or inhibitors of guanylyl cyclase (3 μM ODQ), protein kinase A (10 μM H7, 1 μM H89) and various types of K+ channels (10 μM BaCl2, 3 mM tetraethylammonium, 30 nM charybdotoxin, 30 nM iberiotoxin, 300 nM apamine, 10 μM glibenclamide) had only small if any inhibitory effects against C2‐ceramide‐induced vasodilation, but some of them attenuated vasodilation by sodium nitroprusside or isoprenaline. A combination of ODQ and charybdotoxin almost completely abolished C2‐ceramide‐induced vasodilation. A second administration of C2‐ceramide caused a detectable but weaker relaxation. L‐threo‐C2‐ceramide (100 μM), which should not be a substrate to ceramide metabolism, had no biphasic time course. The ceramidase inhibitor (1S,2R)‐D‐erythro‐2‐(N‐myristoylamino)‐1‐phenyl‐1‐propanol (100 μM) alone caused some vasodilation, indicating vasodilation by endogenous ceramides, and also hastened relaxation by exogenous C2‐ceramide. The late‐developing reversal of C2‐ceramide‐induced vasodilation was absent when α‐adrenergic tone was removed by addition of 10 μM phentolamine. We conclude that C2‐ceramide relaxes rat resistance vessels in an endothelium‐independent manner which is prevented only by combined inhibition of guanylyl cyclase and charybdotoxin‐sensitive K+ channels. The vasodilation abates with time partly due to desensitization of the ceramide response and partly due to metabolism of C2‐ceramide to an inactive metabolite.

Human urinary bladder strip relaxation by the β-adrenoceptor agonist isoprenaline: methodological considerations and effects of gender and age

The present study was primarily designed to explore various methodological aspects related to organ bath experiments evaluating human detrusor relaxation by the β-adrenoceptor agonist isoprenaline. Data are based upon a series of 30 consecutive patients, and this cohort was also used to explore possible effects of gender and age. KCl-induced contraction was related to strip length but not weight or cross-sectional area, indicating that the former is most suitable for data normalization. Storage of detrusor strips in cold buffer for up to 2 days did not affect contractile responses to KCl or efficacy of isoprenaline to cause relaxation but significantly affected the isoprenaline potency. No such alterations were observed with up to 1 day of cold storage. The type (KCl vs. passive tension) or strength of contractile stimulus had only minor effects on isoprenaline responses although these differences reached statistical significance in some cases. Similarly, gender and age had only minor if any effects on KCl-induced contraction or isoprenaline-induced relaxation, but the current data are too limited for robust conclusions. In summary we have evaluated experimental conditions for the testing of human detrusor strip contraction and relaxation which should be useful for future larger studies.

Modulation of noradrenaline-induced microvascular constriction by protein kinase inhibitors.

Abstract. We have tested the role of various protein kinases in noradrenaline-induced, α1A-adrenoceptor-mediated constriction of mesenteric and intrarenal rat microvessels. The protein kinase C inhibitors, H7 and staurosporine, inhibited constriction in both vessel types in concentrations which also inhibit myosin light chain kinase. The more selective protein kinase C inhibitors, bisindolylmaleimide I and Gö 6976, did not inhibit microvessel constriction in concentrations selective for protein kinase C. Moreover, the protein kinase C-activating phorbol ester, phorbol-12-myristate-13-acetate, did not cause constriction. The tyrosine kinase inhibitors, genistein and tyrphostin 23, inhibited constriction in concentrations compatible with tyrosine kinase inhibition. An inhibitor of the extracellular signal-regulated kinase cascade, PD 98059, also caused concentration-dependent inhibition. While chelation of extracellular Ca2+ abolished noradrenaline-induced constrictions, the Ca2+-ATPase inhibitor, thapsigargin, had no effects. We conclude that tyrosine kinases and extracellular signal-regulated kinase (but not protein kinase C) may be involved in noradrenaline-induced rat mesenteric and intrarenal microvessel constriction but this appears to occur independently of an effect on sarcoplasmic Ca2+ storage.