Differences in contractile activation between human myometrium and intramyometrial arteries

Small intramyometrial arteries and pieces of adjacent myometrial tissue were obtained from 25 non-pregnant women undergoing hysterectomy. Vascular and myometrial preparations were dissected, mounted in organ baths and isometric tension was recorded. Myometrial strips, but no vascular preparation, developed spontaneous contractile activity. Noradrenaline (NA) and vasopressin (VP) contracted both vessels and myometrium. Prostaglandin F_2α (PGF_2α) contracted the myometrial tissue, but had only a minor effect on the vessels. Removal of extracellular calcium almost abolished the myometrial responses to high K⁺ (124 mm)-solution, PGF_2α, NA and VP. The vascular responses remaining after this treatment were 18% (K⁺), 34% (NA) and 25% (VP) of control contractions induced by high K⁺ (124 mm). Nifedipine potently depressed myometrial contractions induced by NA and VP, but was less active against the vascular responses to these agents. In preparations exposed to calcium-free medium, nifedipine (10^-7 m) almost abolished myometrial contractions induced by calcium in the presence of K⁺ (124 mm), NA or VP. It also effectively depressed vascular responses to calcium in the presence of K⁺, but was less active if NA and VP were present. It is suggested that PGF_2α has almost no contractant effect on intramyometrial arteries, and that the activation process in these vessels is much less dependent on extracellular calcium than that of the myometrium.     

Effects of prostanoids on isolated feline cerebral arteries. II. Roles of extra- and intracellular calcium for the prostaglandin F2 alpha-induced contraction

The roles of extra- and intracellular calcium for the contractile effects of PGF2 alpha in the feline basilar artery (BA) were investigated. Comparisons were made with contractions induced by K+ and noradrenaline (NA). Addition of nifedipine to PGF2 alpha- or K+ (124 mM)-contracted arteries resulted in an incomplete relaxation, whereas NA-contracted vessels were completely relaxed. Incubation of the preparations in a calcium-free medium containing 10(-5) M EGTA for 5-10 min almost abolished contractions induced by K+ and NA. In contrast, 63% of the response to PGF2 alpha remained after pretreatment of the arteries in a calcium-free solution for 40 min; PGF2 alpha produced a biphasic contraction in 17 out of 20 preparations consisting of a rapidly developing initial phase followed by a second increase in tension after 1-6 min. The second phase was absent if the EGTA-concentration was increased to 10(-4) M, or if the arteries were pre-treated with nifedipine. After incubation of the arteries in a calcium-free medium for 40-120 min and K+-depolarization, re-addition of calcium elicited contractions at lower concentrations in the presence of PGF2 alpha than in controls. The results suggest that PGF2 alpha-induced contractions in the feline BA are considerably less dependent on extracellular calcium than contractions evoked by K+ or NA. PGF2 alpha appears to be able to release calcium from two cellular stores, and may also promote calcium influx through the cell membrane.     

Characterization of the prostanoid receptors and of the contractile effects of prostaglandin F2a in human pial arteries

The contractile and relaxant effects of various prostanoids were studied on isolated human pial arteries. Contractions were elicited with the following order of potency: U46619?U44069>PGB₂>PGF_2a>PGE₂?PGD₂>PGF_1a≥TXB₂, indicating that prostanoid-induced contractions probably are mediated by a thromboxane-sensitive receptor. Relaxation of PGF_2a-contracted arteries was induced with the order of potency: PGE₂> PGE₁>PGD₂?PGD₁. Vessels contrated by K⁺ were relaxed only by PGE,. Since PGI₂ was previously found to be more potent than all the prostanoids tested in the present study, relaxant responses are probably mediated via a PGI₂-sensitive receptor. The roles of free extracellular and cellularly bound calcium for the contractile effects of PGF_2a and K⁺ were estimated by incubating the arteries for various times in calcium-free medium containing 10^-5 M EGTA. Incubation for 5–10 min abolished K⁺-induced contractions, whereas after 40 min of incubation PGF_2a still induced contractions that reached 70% of control. The PGF_2a-induced contraction was biphasic in 8 out of 10 preparations. The second phase could be eliminated by increasing the EGTA-concentration to 10^-4 M, as well as by nifedipine pretreatment. In calcium-free, high K⁺ medium calcium-induced contractions were elicited at lower concentrations in the presence of PGF_2a. The results suggest that PGF_2a-induced contractions in human pial arteries are relatively independent of free extracellular calcium. PGF_2a may promote trans-membrane influx of calcium, as well as release calcium from seemingly superficially located cellular stores.     

Effects of prostanoids on isolated feline cerebral arteries

The effects of various prostanoids on isolated feline basilar arteries (BA) were studied. Contractions were induced with the following order of potency: U 46619 ? U44069 > PGB₂ > PGF_2a? PGA, ? PGB, ≥ PGA, ? PGE₂= PGD₂ > PGF_1a? TXB₂? PGE₁? PGD₁. Distinct bimodal responses with a relaxation at low concentrations followed by a contraction at high concentrations, were induced by PGA₁, PGA₂, PGD, and PGE₂. None of the tested prostanoids relaxed K⁺-contracted arteries, and a sizable relaxant effect in PGF_2a-contracted arteries could be induced only by PGE₁. As judged by the relative order of potency, PG-induced contractions of the feline BA seem to be mediated by a thromboxane sensitive receptor. PGF_2a-induced contractions apparently do not involve the release of noradrenaline from perivascular nerves since phentolamine failed to affect contractions induced by this agent.     

Influence of extracellular calcium and nifedipine on α1-and α2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries

The influence of extracellular Ca²⁺ and nifedipine on contractile responses to 10 μM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly α₂-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly α₁-adrenoceptors) and CMA (sensitive to both at,- and α₂-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca²⁺-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K⁺ was more sensitive to Ca²⁺ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca²⁺, can also release Ca²⁺ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, α₁-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca²⁺ influx and intracellular Ca²⁺ release, whereas α₂-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca²⁺ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca²⁺-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca²⁺, whereas in the other types of artery, Ca²⁺ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca²⁺ influx and intracellular Ca²⁺ release to the contractile activation, but also in the nifedipine sensitivity of the Ca²⁺ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of α-adrenoceptor stimulated by NA and to the type of vessel studied.     

Vasodilator responses to alpha-human-atrial natriuretic peptide in isolated omental and pulmonary arteries from rabbit and man

In isolated vessels from the pulmonary and mesenteric/omental arterial circulations of rabbit and man, the effects of synthetic α-human atrial natriuretic peptide (α-hANP) were investigated. The vessels had an outer diameter of 0.6–1.2 mm and were contracted by noradrenaline (NA) and prostaglandin (PG) F_2α, (mesenteric/omental arteries) or by 5–hydroxytryptamine (5–HT) and PGF_2α (pulmonary arteries). It was found that in rabbits, mesenteric vessels contracted by NA and PGF_2α were not significantly relaxed by α-hANP. Rabbit pulmonary vessels contracted by PGF_2α or 5–HT were concentration-dependently relaxed by α-hANP (maximum 70%). Human omental vessels contracted by NA and PGF_2α showed a moderate (30%) relaxation after addition of the peptide. Human pulmonary arteries contracted by 5–HT were relaxed by α-hANP almost to baseline, while PGF_2α-contracted vessels showed a maximum relaxation of 65%. It is concluced that α-hANP has a relatively selective effect on pulmonary arterial vessels from rabbit as well as man. The results suggest that the peptide may be involved in the regulation of pulmonary vascular tone.     

Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline

 We evaluated the relationship between intracellular calcium concentration ([Ca²⁺]_i) and vasoconstriction during the presence of exogenous noradrenaline (NA) and sympathetic nerve stimulation. An imaging technique was used to determine calcium/tension relationships in isolated rat mesenteric resistance arteries that had been mounted for recording of isometric tension development and loaded with Fura-2/AM. Experiments were performed after depletion of vasodilator neuropeptides and in the continuous presence of 1 μM propranolol, 3 μM indomethacin, and 30 μM nitro-l-arginine. NA (10 μM) was shown first to induce a further increase in tension, but not [Ca²⁺]_i, during the contraction induced by 125 mM K⁺. Subsequently, calcium/tension relationships were determined during stimulation with graded increases in extracellular [K⁺] (5.9–125 mM K⁺), cumulative administration of NA (0.2–10 μM) and electrical field stimulation of perivascular nerves (EFS, 1–16 Hz). A basal calcium/tension relationship without the calcium-sensitizing property of NA was constructed using a cumulative concentration/response curve of 5.9–125 mM K⁺ in arteries after prior exposure to the irreversible α-adrenoceptor antagonist phenoxybenzamine (POB). K⁺ series before and during α-blockade were also studied using the combination of the α₁-antagonist prazosin and α₂-antagonist yohimbine yielding comparable results as with POB. Calcium/tension curves obtained in the presence of NA, K⁺ and during EFS all were shifted to the left compared with the basal condition and all showed a similar slope indicating that neurogenically released NA is equally capable of inducing calcium sensitization in smooth muscle of mesenteric resistance arteries as exogenously applied NA. In the presence of exogenous and endogenous NA we not only observed an elevated contractile response for a given increase in [Ca²⁺]_i, but also an attenuated rise in [Ca²⁺]_i for a given intensity of stimulation. This suggests that the agonist-induced calcium-sensitization is accompanied by a reduction of the rise in [Ca²⁺]_i. Received: 24 October 1997 / Received after revision: 26 January 1998 / Accepted: 26 February 1998     

Enhancement of depolarization-induced contractions after endothelium denudation is not related to an impaired production of nitric oxide or prostacyclin in the rabbit basilar artery

Enhancement of the extracellular potassium ion (K⁺) concentration combined with endothelial injury have been suggested to occur during cerebral ischaemia-reperfusion and vasospasm after subarachnoid hemorrhage. The effect of potassium (K⁺) depolarization was therefore investigated in isolated segments of the rabbit basilar artery with and without an intact endothelial cell layer. Addition of potassium chloride to the organ bath induced a concentration-dependent contraction. Endothelial denudation of the artery resulted in an unstable baseline tension and a leftward shift of the K⁺ concentration-response curve. The K⁺ concentration eliciting half maximum contraction decreased from 26 mmol l^-1 in the presence to 12 mmol l“¹ in the absence of an intact endothelium. Nimodipine (3 × 10^-7 mol l^-1) or exposure to a calcium-free medium abolished the spontaneous as well as K⁺-induced contractions. A^-nitro-L-arginine (10^-4 mol l^-1), indomethacin (3 × 10^-6 mol l^-1) and glibenclamide (10^-5 mol I^-1) did not affect the contractile response to K⁺ in intact arteries. However, N_w-nitro-D-arginine increased the baseline tension, and this effect could not be reproduced with N_w-nitro-D-arginine. Pinacidil (10^-6mol l^-l) abolished the spontaneous contractile activity in endothelium-denuded arteries and reduce the K⁺ sensitivity to the same level as in intact arteries. Tetraethylammonium (3 mmol l^-1) and ouabain (10^-5 mol I^-l) increased the basal tension and shifted the K⁺ concentration-response curve to the left. Calcium-induced contractions in preparations exposed to a calcium-free, 124 mmol l^-l K.⁺ solution did not differ between endothelium-denuded and intact arteries. It is suggested that the endothelium of the rabbit basilar artery releases a hyperpolarizing factor, distinct from nitric oxide or a cyclooxygenase product, which attenuates the vasoconstrictor effect of K⁺ depolarization.     

Endogenous and exogenous agonist-induced changes in the coupling between [Ca2+]i and force in rat resistance arteries

The relationship between isometric tension and free cytoplasmic calcium, [Ca²⁺]_i, was investigated in rat isolated resistance arteries using fura-2. Depolarisation with 125 mM K⁺ induced a tonic contraction, while [Ca²⁺]_i increased transiently but stabilised above resting [Ca²⁺]_i. Furthermore, the tension/[Ca²⁺]_i ratio was lower during activation with 125 mM K⁺ if the effect of endogenous noradrenaline (NA) was inhibited. Concentration/ response curves with NA and K⁺ indicated that NA increased the sensitivity to [Ca²⁺]_i. Calcium concentration/response curves in the presence of 10 M NA or 125 mM K⁺ showed that NA could induce force at or below resting [Ca²⁺]_i, while for any given bath calcium concentration, [Ca²⁺]_i was similar in the presence of NA or K⁺. Addition of NA or vasopressin (AVP) to vessels depolarised with 125 mM K⁺ caused force development but no increase in [Ca²⁺]_i, suggesting that agonists increase the efficacy of [Ca²⁺]_i. However, during activation with AVP the efficacy of [Ca²⁺]_i decreased time-dependently. The results suggest that in resistance arteries [Ca²⁺]_i plays a crucial role in excitation-contraction coupling, but the tension/[Ca²⁺]_i relationship can be modified by exogenous and endogenous agonists.     

Effects of prostaglandin E1, E2, and F2α on isolated pial arteries of cat

Responses to prostaglandin (PG), E₁, E₂, and F_2α were studied on isolated feline middle cerebral arteries. At resting state PGF_2α produced strong dose-dependent contractions. PGE₂ elicited weak relaxations at low concentrations, followed by powerful contractions at higher doses. PGE₁ had little effect on resting pial vessels. The relative constrictory potency was PGF_2α > PGE₂ > PGE₁. During active tone, induced by administration of either potassium, norepinephrine, or 5-hydroxytryptamine, relaxations induced by PGE₁ were enhanced, whereas PGE₂-induced relaxations were unaffected. PGE₁ induced relaxations were more pronounced when the active tension had been produced by administration of PGF_2α than with either of the vasoactive amines or potassium. This study demonstrates the importance of smooth muscle tone, and by what means this is achieved, when examining the responses of PG's on cerebral blood vessels.     

Neuropeptide Y regulates rat renal tubular Na,K-ATPase through several signalling pathways

Neuropeptide Y (NPY) has at least three receptors (Y₁, Y₂ and Y₃) through which it influences different mechanisms in many cell types. Previous data suggest that the Y₂ receptor may be divided into prejunctional and postjunctional subgroups. We have examined the intracellular signalling pathways of the postjunctional Y₂ receptor in rat renal proximal tubules. The results indicate that NPY regulates Na⁺,K⁺-ATPase through several signalling pathways: (1) In proximal tubule (PT) cells NPY increased intracellular calcium. The response was blocked by removing extracellular calcium and was also blocked by using nifedipine. This suggests that calcium was increased by influx from the extracellular space through L -type calcium channels. (2) NPY increased Na⁺,K⁺-ATPase activity in PT segments and this effect was also blocked by nifedipine. CaMKII-Ala²⁸⁶[281–302] a blocker of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibited the NPY-stimulated Na⁺,K⁺-ATPase activity. This implies that increased intracellular calcium activates CaMKII which subsequently increases Na⁺,K⁺-ATPase activity. CaMKII thus appear to act similar to what has been proposed for protein phosphatase 2B. (3) Calphostin C, an inhibitor of protein kinase C (PKC), did not inhibit NPY-stimulated Na⁺,K⁺-ATPase activity. PKC is, therefore, unlikely to be involved. (4) Y₂ receptors are negatively coupled to the cAMP pathway. NPY attenuated forskolin-stimulated cAMP production in renal tubules and exogenous cAMP counteracted the NPY-stimulated Na⁺,K⁺-ATPase activity. This illustrated the importance of NPY for the regulation of renal sodium handling. We also propose that the renal tubule cell is a good model for studying the function and mechanisms of postjunctional Y₂ receptors.     

A new view of K+ -induced contraction in rat aorta: the role of Ca2+ binding

Strong, K⁺-induced contractions of rat aorta in Ca-free, Mg-free media were not accompanied by increased intracellular calcium concentration, [Ca²⁺]_i, whereas such contractions in the presence of the divalent cations were correlated with rising [Ca²⁺]_i as assessed by fura-2. At the same time, calcium channel blockers, a modulator of Ca²⁺-binding proteins, and a modulator of actin polymerization, inhibited all types of K⁺-induced contractions. Increasing the K⁺ in isotonic medium evoked a rise of ⁴⁵Ca²⁺ binding to the plasma membrane of freshly isolated aortic cells. Although Ca²⁺-dependent events underlie the mechanism of K⁺-induced vascular contractions in both the presence and absence of Ca²⁺, in contrast to the view that [Ca²⁺]_i is a key regulator of excitation-contraction coupling in smooth muscle, we suggest that the modulation of Mg²⁺-dependent Ca²⁺ binding, probably within/at the L-type calcium channel by K⁺, is a trigger for aortic contraction. This Ca²⁺ binding may then activate actin-myosin interaction.     

Effect of verapamil on restoration of cardiac performance in raised [K+]0 by adrenergic stimulation in the rabbit

Modulation of the L-type calcium channel by catecholamines improves action potential parameters in single ventricular myocytes depolarized by high [K⁺]₀ Tyrode. Whether this modulation is important in offsetting the negative effects of hyperkalaemia in the whole heart is not known. We tested the effects of the calcium channel antagonist, verapamil, on restoration of cardiac performance by adrenergic stimulation in high [K⁺]₀ in anaesthetized rabbits and isolated perfused working rabbit hearts. Raised [K⁺]₀ decreased SBP, LVP and LVdP/dt_maxin vivo ([K⁺]_a 8.6 ± 0.2 mM; n= 10) and aortic flow (AF) in the isolated heart (8 mM [K⁺]₀ Tyrode; n= 25). However, the negative effects of raised [K⁺]_a were offset by isoprenaline (Iso, 1 μg kg^-1 min^-1 i.v.) in vivo and by noradrenaline (NA, 80 nM) in the isolated heart. Verapamil (0.15 mg kg^-1 iv.; 15 nM isolated heart) markedly potentiated the negative inotropic effects of raised [K⁺]_n in both preparations. Verapamil attenuated the effect of isoprenaline in vivo but in the isolated heart, the protective effect of NA in 8 mM [K⁺] Tyrode (AF 97 ± 10 mL min¹ in 8 mM [K⁺]₀ compared with AF 141 ± 8.5 mL min^-1 in 8 mM [K⁺]₀+ NA) was offset by the drug (90±8mL min^-1 in 8 mM [K⁺]₀+ NA + V). Furthermore, verapamil abolished aortic flow in 8 mM [K⁺]₀ alone. These findings suggest that the heart may be critically dependent on modulation of intracellular calcium in order to tolerate concentrations of K⁴ similar to those seen during a short burst of intensive exercise ([K⁺]_a 8.6 mM).     

Effects of stimulation and inhibition of protein kinase C on the cytosolic calcium concentration in rabbit afferent arterioles

The effect of the protein kinase C (PKC) inhibitor chelerytrine (Ch) and the PKC activator 12–0-tetradecanoyl-phorbol-13-acetate (TPA) on the cytosolic calcium concentration ([ Ca²⁺]_i) in isolated intact rabbit afferent arterioles was investigated. [Ca²⁺]_i was measured in the proximal and distal parts of the arteriole. Administration of 1 μM Ch gave rise to a peak followed by an elevated level of [Ca²⁺]_i in both these parts. Neither the peak nor the elevated level of [Ca²⁺]_i was significantly reduced by 1 μM nifedipine. The relative peak increase in [Ca²⁺]_i in response to 1 μM noradrenaline (NA) or to 10 nM angiotensin II (AII) was significantly blunted in both parts after preincubation with 1 μM Ch. Depolarization with 25 mM K⁺ increased [Ca²⁺]_i in both parts. Preincubation with Ch did not affect the increase in [Ca²⁺]_i induced by 25 mM K⁺. TPA (10 and 100 nM ) did not significantly affect the basal [Ca²⁺]_i in the afferent arteriole. The [Ca²⁺]_i response to NA or 25 mM K⁺ was not affected by TPA. We conclude that blockade of PKC increases [Ca²⁺]_i in afferent arteriolar smooth muscle by a mechanism independent of L-type voltage-sensitive calcium channels. Inhibition of PKC blunts the relative increase in [Ca²⁺]_i in response to AII and, to a lesser extent, that induced by NA. We conclude that PKC might be important in modulating the calcium changes that occur in response to these vasoconstrictors.     

Effects of Prostaglandins on the Isolated Human Bladder and Urethra

The effects of prostaglandins F_2α (PGF_2α), E₂ (PGE₁) and E₂ (PGE₂) on the human lower urinary tract were studied in vitro in preparations obtained from patients undergoing total cystourethrectomy because of bladder malignancy. Tissue specimens were taken from different parts of the urethra, the urethrovesical junction, and the bladder. From these specimens, smooth muscle preparations were dissected and mounted in organ baths, that were filled with Krebs solution (37°C) and bubbled with carbogen. Isometric tension was recorded. Preparations from the bladder and all parts of the urethra were contracted by PGF_2α. This effect was not affected by tetrodotoxin, phenoxybenzamine, or atropine; isoprenaline relaxed the PGF_2α induced contractions. PGE₁ and PGE₂ both contracted strips from the bladder. However, urethral preparations contracted by PGF_2α or noradrenaline were relaxed by these agents. This relaxing effect was at least as pronounced as that produced by isoprenaline; it was not affected by propranolol.     

Contractile effects of endothelin-I and localization of endothelin binding sites in rabbit lower urinary tract smooth muscle

In isolated rabbit bladder and urethral smooth muscle, endothelin-1 caused concentration-related, slowly developing contractions that were difficult to wash out. Relative to contractions induced by K⁺ (124 mM), contractions in bladder preparations reached a higher amplitude than in urethral preparations. There was a marked tachyphylaxis to the effects of the peptide. The endothelin-l-induced contractions were not significantly affected by phentolamine or indomethacin in the urethra, or by scopolamine or indomethacin in the bladder. Incubation for 30 min in a Ca²⁺-free solution abolished the endothelin-l-induced contractions. Nifedipine did not affect the actions of endothelin-1 in the urethra but had a marked inhibitory action on its effects in the bladder. In the presence of endothelin-1, Ca²⁺-induced contractions were significantly blocked by nifedipine in the bladder but not in the urethra. Urethral preparations at resting tension responded to electrical stimulation by tetrodotoxin-sensitive, frequency-dependent contractions sensitive to α-adrenoceptor blockade. Pretreatment with endothelin-1 (10^-9′ M) produced a significant increase in the nerve-induced contractions but had no significant effect on contractions induced by exogenous noradrenaline. Endothelin-1 did not affect spontaneous or stimulation-induced efflux of ³H-labelled noradrenaline in urethral smooth muscle. Preparations contracted by endothelin-1 were frequency-dependently relaxed by electrical stimulation. The peptide had no significant effect on the responses induced by electrical stimulation in the bladder preparations. In both bladder and urethra, [¹²⁵]endothelin-l binding sites were found mainly in the outer longitudinal muscle layer, in vessels and in the submucosa. The highest density of binding sites appeared to be in vessels and the outer muscle layer in both types of muscle. The results suggest that in the rabbit both bladder and urethral smooth muscle contain binding sites for endothelin. The peptide has contractant effects dependent on extracellular calcium in both types of tissue, but voltage-operated calcium channels seem to involved in activation only of bladder smooth muscle. The functional importance of endothelin-1 in the rabbit lower urinary tract remains to be elucidated.     

Conversion of arachidonic acid to prostaglandins in homogenates of human skeletal muscle and kidney

The capacity of human skeletal muscle and kidney homogenates to synthetize prostaglandins (PGs) from exogenous precursor was investigated. Low–speed supernatants of muscle as well as renal medullary and cortical homogenates were incubated with ¹⁴C–labelled arachidonic acid (¹⁴C–AA) prepared as a sodium salt. ¹⁴C–PGs in the incubates were extracted, separated with thin–layer chromatography (TLC) and quantified by radioscanning. In the skeletal muscle incubates ¹⁴C–AA was converted into ¹⁴C–PGs with a time–dependent yield, most effectively after 10–15 min incubation. Well–defined radiopeaks parallel to unlabelled standards of PGD₂, PGE₂, PGF_2α and 6–keto–PGF_1α were obtained in the chromatograms. PGE₂ was the main PG formed, constituting over 50% of ¹⁴C–activity, whereas 6–keto–PGF_1α, PGD₂ and PGF_2α were found in considerably lower proportions. In the renal medullary incubates, PGE₂ likewise accounted for the largest part of ¹⁴C–PGs formed, but significant relative amounts of PGF_2α and PGD₂ were also found. A minor peak, corresponding to 6–keto–PGF,_a and thus indicating formation of PGI₂, was also obtained. In contrast to the medulla, no ¹⁴C–PGs could be found in the renal cortical incubates. The results demonstrate the existence of a considerable tissue specificity in the quantitative and qualitative expression of PG biosynthesis in man.     

Role of prostaglandins in lipopolysaccharide effects on K+‐induced contractions in rabbit small intestine

Aim: The mediators of the pathophysiologcal symptoms of septic shock are not completely understood. The aim of this work was to investigate the effect of lipopolysaccharide (LPS) on the K⁺‐induced response of longitudinal segments of rabbit small intestine in vitro and the possible role of prostaglandins. Methods and results: Rabbits were treated with intravenously injected LPS. After 90 min animals were killed and intestinal segments were mounted in an organ bath. Lipopolysaccharide (0.2 μg kg^?1) inhibited K⁺‐induced contractions (60 mm ) by 68% in duodenum, 58% in jejunum and 52% in ileum. Indomethacin antagonized LPS actions when injected 15 min before LPS. PGE₂ reduced K⁺‐induced contractions, imitating LPS effects. In contrast, contractions induced by K⁺ increased when intestinal segments were incubated in vitro with LPS for 90 min. The LPS (0.3 μg mL^?1) increased K⁺‐induced contractions (60 mm ) by 46% in duodenum, 63% in jejunum and 85% in ileum. The LPS effect was antagonized by indomethacin at 10^?6 m in duodenum and jejunum and at 10^?8 m in ileum. PGE₂ evoked dose‐dependent contractions when added to the bath in duodenum, jejunum and ileum. Conclusion: These results suggest that effect of LPS on K⁺‐induced contractions in the rabbit small bowel may be mediated by prostaglandin E₂.     

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.     

Effect of catecholamines on the ventricular myocyte action potential in raised extracellular potassium

We describe the relationship between catecholamines and raised extracellular potassium ([K⁺]_o) on action potential parameters and calcium currents in isolated ventricular myocytes of the guinea-pig and relate these findings to the problem of understanding how the heart is protected from exercise-induced hyperkalaemia ([K⁺]_a up to 8.5 mm ). Action potential duration (APD₉₀), amplitude and upstroke velocity were recorded in stimulated (2Hz) guinea-pig ventricular myocytes using whole-cell patch electrode recordings (37 ±C). Cells were superfused with normal K⁺Tyrode and with raised K⁺Tyrode in the presence of either noradrenaline, adrenaline or raised calcium. Inward calcium current was measured using voltage clamp. Raised K⁺(8, 12, 16 mm K⁺Tyrode) caused a significant (P < 0.01) depolarisation, shortened the APD₉₀ and decreased the action potential amplitude and upstroke velocity. In raised K⁺Tyrode addition of noradrenaline (0.08–0.1 μm ) or adrenaline (0.1–0.2 μm ) increased action potential amplitude (P < 0.01), APD₉₀ (P < 0.01) and upstroke velocity (P < 0.01) (measured only in 16 mm K⁺Tyrode). In 12 mm K⁺Tyrode raised Ca²⁺(5–6 mm ) increased action potential amplitude (P < 0.05) and shortened APD₉₀ (P < 0.05). Addition of NA (0.08–0.1 μm ) increased the inward Ca²⁺current. All effects were fully reversible. In raised [K⁺]_o increases in catecholamines and [Ca²⁺]_o cause changes in action potential parameters that would be expected to maintain propagation of the cardiac action potential in the whole heart. Thus, in the ventricular myocyte the increase in conductance to Ca²⁺caused by catecholamines may be one factor that is important in minimising the potentially adverse effects of exercise-induced hyperkalaemia.