ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

EFFECTS OF ENDOTHELIUM-DERIVED RELAXING FACTOR ON THE SMOOTH MUSCLE OF THE RAT TAIL ARTERY

Simultaneous measurements of smooth muscle membrane potential and tension were made from isolated pieces of rat tail artery. A single electrical stimulus to the perivascular nerves produced a transient contraction of the smooth muscle. The amplitude of the contraction was increased after removal of the endothelium. The excitatory junction potentials and action potentials in the smooth muscle had the same amplitudes before and after removal of the endothelium. Tension obtained by direct stimulation of the arterial muscle in guanethidine-treated arteries was also increased by removal of the endothelium. When the artery was constricted by noradrenaline or 5-hydroxytryptamine, electrical stimulation caused a relaxation that was reduced by removing the endothelium. It was concluded that the electrical stimulus released the endothelium-derived relaxing factor (EDRF) which reduced the amount of contractile force that could be produced by an action potential in the smooth muscle.     

RELAXANT EFFECTS OF BRAIN NATRIURETIC PEPTIDE ON GUINEA-PIG TRACHEAL SMOOTH MUSCLE

1. The relaxant effects of brain natriuretic peptide (BNP) were investigated on guinea-pig tracheal smooth muscle. 2. Various amounts of BNP (10^?9– 10^?6 mol/L) showed concentration-dependent relaxant effects on resting tone, leukotriene D₄ (LTD₄; 10^?8 mol/L) and endothelin-1 (ET-1; 10^?8 mol/L) induced contraction of tracheal smooth muscle with EC₅₀ values of 3.1± 0.7 ± 10^?8, 3.9 ± 1.0 ± 10^?8 and 3.5 ± 1.0 ± 10^?8 mol/L, respectively. 3. BNP increased tissue cyclic GMP levels in tracheal smooth muscle concentration dependently (187 ± 26 fmol/mg protein in control, 334 ± 77 fmol/mg protein at 10^?8 mol/L, 680 ± 54 fmol/mg protein at 10^?7 mol/L, 2162 ± 133 fmol/mg protein at 10^?6 mol/L). 4. With the addition of BNP, tissue cyclic GMP levels reached a maximum at 1–3 min. The relaxation of tracheal smooth muscle began at 1 min and reached a maximum level at 5 min after the superfusion of BNP (10^?6 mol/L). The elevation of cyclic GMP preceded the relaxation of tracheal smooth muscle. 5. These results suggest that BNP may have a potent relaxant effect on tracheal smooth muscle and this effect may be mediated by cyclic GMP level.     

EFFECTS OF RENAL DENERVATION AND ATRIAL NATRIURETIC FACTOR ON TUBULAR REABSORPTION IN ANAESTHETIZED RATS

1. The effects of acute unilateral renal denervation were examined in 17 anaesthetized rats. Renal haemodynamic changes were monitored using standard clearance techniques. Lithium clearance was used to assess fractional proximal sodium and water reabsorption. 2. Denervation resulted in ipsilateral renal vasodilatation with marked natriuresis and diuresis, a small increase (15%, P less than 0.05) in glomerular filtration rate (GFR) and a consequent reduction in filtration fraction. Fractional lithium reabsorption decreased (67.3 +/- 2.9% to 54.5 +/- 4.0%, P less than 0.01) and absolute proximal reabsorption did not change, indicating impairment of proximal glomerulotubular balance (GTB). No similar changes in haemodynamic or transport parameters were observed in the contralateral, innervated kidney, although vascular resistance increased. 3. In 9 experiments following denervation of the left kidney, systemic low dose infusion (10 ng/min) of atrial natriuretic factor (ANF) resulted in a fall in mean arterial blood pressure from 116 +/- 3 mmHg to 107 +/- 3 mmHg (P less than 0.05). In the denervated kidney ANF increased urine flow rate and sodium excretion to rates above those established following denervation alone. However, in the right kidney, despite the increased filtered load (35%, P less than 0.01), the natriuretic and diuretic responses to ANF were abolished. 4. In the denervated kidney, ANF further reduced the fractional reabsorption of lithium from 53.6 +/- 2.3% to 45.6 +/- 3.8% (P less than 0.05). GFR increased by 32% (a total of 49% higher than during pretreatment) but absolute proximal reabsorption (APR) did not change. However, in the right, innervated kidney ANF infusion produced a 35% increase in GFR accompanied by a 53% rise in APR. 5. It is concluded that the natriuresis induced by unilateral denervation is due predominantly to impaired proximal GTB. The natriuretic action of ANF was associated with further impairment of proximal GTB, not dependent upon decreasing activity of renal sympathetic nerves, but was abolished when filtration fraction and renal sympathetic tone were elevated.     

RENAL EFFECTS OF ATRIAL NATRIURETIC FACTOR (99–126) IN POTASSIUM LOADED SHEEP

1. The renal response to renal arterial infusion of synthetic atrial natriuretic factor (ANF 99-126) was examined in conscious sheep following dietary K loading, and compared with the response in normal sheep. 2. Renal arterial infusion of ANF in K loaded sheep increased the excretion of Na and Ca, but did not affect the excretion of K. 3. The natriuretic effect of ANF was attenuated in K loaded animals, possibly as a consequence of the reduction in Na status which is associated with K loading.     

CIRCULATING Na+/K+-ATPase INHIBITORS: EFFECTS OF NEUROPEPTIDES, VOLUME EXPANSION AND SALT LOADING IN CONSCIOUS RATS

1. In mammalian plasma, many different inhibitors of Na⁺/K⁺-ATPase are present, but it is not clear whether their net effect on Na⁺/K⁺-ATPase activity changes during the regulation of electrolyte and fluid balance. We studied Na⁺/K⁺-ATPase inhibition by plasma extracts in conscious rats during short-and long-term body fluid regulation. 2. Male, adult, conscious, freely moving Wistar rats were subjected to one of the following protocols: (i) intracerebroventricular (i.c.v.) injections of angiotensin II (Angll; 1, 10 and 100 ng), the Angll receptor antagonist losartan (1μg), atrial natriuretic peptide (ANP-III; 1μg) or isotonic saline (IS); (ii) intra-arterial (i.a.) injections of IS (6 or 10 mL), hypertonic saline (HS; 1.2% NaCl, 5 mL) or hypertonic plasma expander (HPS; 3.5% hetastarch in HS, 5mL); or (iii) a low salt-high salt-low salt diet sequence (0.18/1.8/0.18% NaCl chow for S days each with controls receiving 0.18% NaCl on all days). Bodyweight, the intake of food and water, urine volume and Na⁺ concentration and weight of faeces were determined daily. Plasma samples were withdrawn repeatedly throughout the respective protocols, extracted on C18-reversed phase columns and assayed for their effect on the activity of different Na⁺/K⁺-ATPase preparations. 3. The inhibition of rat brain Na⁺/K⁺-ATPase by plasma extracts was not significantly changed by i.c.v. injection of Angll, losartan, ANP-III and IS within the observation period (30 min from respective stimuli). Similarly, no significant changes occurred after acute volume expansion by i.a. injection of IS or HS within 120 min; upon HPS, however, Na⁺/K⁺-ATPase inhibition was decreased by approximately 20% (P<0.05), probably due to passive dilution. During the high-salt diet, fluid retention was effectively counteracted by an adaptive increase of urinary sodium excretion. Throughout the protocol, inhibition of pig brain Na⁺/K⁺-ATPase by plasma extracts did not differ significantly between groups. 4. It is concluded from these results that the short- or long-term control of body fluids in conscious rats is not associated with systematic changes in Na⁺/K⁺-ATPase inhibition by plasma factors.     

STUDIES ON AGE-RELATED CHANGES IN VASCULAR SMOOTH MUSCLE AND THEIR Ca2+ MECHANISMS

1. Increases in cell proliferation and DNA synthesis were observed in vascular smooth muscle cells (VSMC) from old rats. These effects were significantly enhanced by noradrenaline but were inhibited by nifedipine. 2. Ginsenosides, traditional Chinese drugs, inhibited the proliferation and DNA synthesis in VSMC from old rats. Cytosolic and nuclear Ca2+ levels, as well as calmodulin activity, were clearly higher in old rats compared with young rats. 3. Both Ca2+ and calmodulin levels rose abruptly in the late G1 phase in the VSMC cell cycle in old rats. 4. The increase in inositol phosphate levels stimulated by phenylephrine in VSMC was greater in old than in young rats. 5. The platelet-derived growth factor (PDGF) gene was overexpressed in old rats. The results indicate that vascular ageing is related to enhanced proliferation of VSMC. Abnormal Ca2+ homeostasis as well as overexpression of the PDGF gene may be responsible. 6. Nifedipine and ginsenosides may inhibit VSMC proliferation with age.     

FORCE AND INTRACELLULAR Ca2+ DURING NANC-MEDIATED RELAXATION OF RAT ANOCOCCYGEUS MUSCLE AND THE EFFECTS OF CYCLOPIAZONIC ACID

1. Simultaneous recordings of tension and [Ca²⁺]_i during NANC-mediated relaxation were made in the rat anococcygeus muscle under various conditions. 2. In muscles precontracted with guanethidine, nitrergic stimulations at 2 Hz produced a rapid decrease in both the tension and [Ca²⁺]_i. 3. The nitric oxide synthase inhibitor, N^G-nitro-L-Arginine (NOLA, 100 μmol/L) completely abolished the decreases in the [Ca²⁺]_i and force response of the NANC-mediated relaxation. 4. Noradrenergic-mediated contractions elicited by electrical field stimulation were potentiated by the addition of NOLA. In the absence of NOLA, the motor responses were larger in magnitude at 10 Hz stimulation than at 2 Hz. After NOLA, both the force response and the associated rise in [Ca²⁺]_i were substantially increased in comparison to the control stimulations. Proportionately the potentiation of the 2 Hz response was of a far greater magnitude than that of the 10 Hz response. 5. The guanylate cyclase inhibitor methylene blue (10 μmol/ L), partially inhibited the force and [Ca²⁺]_i response of the NANC relaxation. 6. Following exposure of the muscles to the sarcoplasmic reticulum Ca²⁺-ATPase inhibitor, cyclopiazonic acid, (10 μmol/ L) the responses to NANC stimulation were inhibited. The attenuated relaxation response displayed a bi-phasic timecourse and the Ca²⁺ change in comparison to that of the control was markedly smaller. In some cases, a relaxation was observed with no detectable change in the [Ca²⁺]_i. 7. The results suggest that part of the relaxation response observed with NANC-mediated relaxation in the rat anococcygeus is dependent on Ca²⁺ sequestration into the sarcoplasmic reticulum. However, other Ca²⁺ lowering mechanisms and possible Ca²⁺ independent mechanisms may also contribute to the NANC relaxation response.     

RELATIVE CONTRIBUTIONS OF EXTRACELLULAR Ca2+ AND Ca2+ STORES TO SMOOTH MUSCLE CONTRACTION IN ARTERIES AND ARTERIOLES OF RAT, GUINEA-PIG DOG AND RABBIT

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca²⁺ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 μmol/L) with a transient contraction in Ca²⁺-free Krebs', reflecting Ca²⁺ release from PE-sensitive Ca²⁺ stores. After pretreatment with cyclopiazonic acid (CPA; 30 μmol/L) or thapsigargin (TSG; 1 μmol/L), putative SR Ca²⁺ pump inhibitors, the PE-induced contraction in a Ca²⁺-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 μmol/L), an agonist that enhances Ca²⁺ release from the SR, also reduced the PE contraction in a Ca²⁺-free solution. 3. CPA or TSG alone in the presence of extracellular Ca²⁺, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca²⁺ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca²⁺ fluxes across the plasma membrane predominates.     

INCREASED Na+/H+ EXCHANGE ACTIVITY IN VASCULAR SMOOTH MUSCLE CELLS OF SPONTANEOUSLY HYPERTENSIVE RATS AND POSSIBLE INVOLVEMENT OF PROTEIN KINASE C

1. Na+ influx into cultured vascular smooth muscle cells (VSMC) obtained from spontaneously hypertensive rats (SHR) and from Wistar-Kyoto rats (WKY) was measured. Na+ influx via the Na+/H+ exchange system was measured as the rate of 22Na+ influx into cultured VSMC sensitive to ethylisopropylamiloride (EIPA), a specific inhibitor of the exchange system. 2. The total 22Na+ influx rate in SHR was significantly higher than in WKY (6.08 +/- 0.16 vs 4.13 +/- 0.09 nmol/min per mg protein; P less than 0.001; n = 14). The EIPA (1 X 10(-4) mol/L)-sensitive 22Na+ influx rate in SHR was significantly higher than that in WKY (4.32 +/- 0.27 vs 2.17 +/- 0.14 nmol/min per mg protein; P less than 0.001; n = 14). There was no difference in EIPA-insensitive 22Na+ influx between SHR and WKY. The EIPA-sensitive 22Na+ influx rate into VSMC was significantly decreased in SHR but not in WKY by the addition of 1 X 10(-4) mol/L 1-(5-isoquinoline-sulfonyl)-methylpiperazine (H-7), an inhibitor of protein kinase C (PK-C). 3. These results suggest that the increase in Na+ influx in SHR may be due to elevation of the Na+/H+ exchange activity, and possible involvement of PK-C in the increased Na+/H+ exchange activity in VSMC from SHR.     

HUMORAL DETERMINANTS OF Na+ EXCRETION AFTER INTRAVENOUS NaCl LOADING IN NORMAL VOLUNTEERS

1. Twelve healthy volunteers maintained on a 100 mmol/day Na⁺ diet, were given an intravenous infusion of 2L saline (0.9%) between 10.00 and 13.00h on 2 study days at least 1 week apart. Urine collections (90 min) were made from 08.30 to 16.00 h. Either carbidopa 100 mg or indomethacin 50 mg was given orally at 07.45 h on one study day and placebo was given on the other (in random order). 2. On the placebo day, saline infusion caused significant decreases in plasma albumin concentration, plasma renin activity (PRA), plasma aldosterone concentration and urinary aldosterone excretion, with 2 to 3-fold increases in plasma atrial natriuretic peptide (ANP) concentration and urinary dopamine: noradrenaline ratio (DA:NA), whereas mean urinary kallikrein and prostaglandin E₂ (PGEI) excretion rates were unchanged. Carbidopa decreased urinary DA:NA and indomethacin decreased urinary PGE₂ excretion, compared with the placebo day. Excretion of sodium (Na⁺) decreased below baseline in two out of six carbidopa-treated subjects and in three out of six indomethacin-treated subjects, but showed little or no change in the remainder. 3. These preliminary observations suggest that some subjects in the early phase of natriuresis after an intravenous Na⁺ load can be identified as having prostaglandin-dependent or dopamine-dependent mechanisms for Na⁺ excretion.     

ROLE OF CYCLIC NUCLEOTIDES IN THE CONTROL OF CYTOSOLIC Ca2+ LEVELS IN VASCULAR ENDOTHELIAL CELLS

• 1 Endothelial cells have a key role in the cardiovascular system. Most endothelial cell functions depend on changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) to some extent and Ca²⁺ signalling acts to link external stimuli with the synthesis and release of regulatory factors in endothelial cells. The [Ca²⁺]_i is maintained by a well‐balanced Ca²⁺ flux across the endoplasmic reticulum and plasma membrane.
• 2 Cyclic nucleotides, such as cAMP and cGMP, are very important second messengers. The cyclic nucleotides can affect [Ca²⁺]_i directly or indirectly (via the actions of protein kinase (PK) A or PKG‐mediated phosphorylation) by regulating Ca²⁺ mobilization and Ca²⁺ influx. Fine‐tuning of [Ca²⁺]_i is also fundamental to protect endothelial cells against damaged caused by the excessive accumulation of Ca²⁺.
• 3 Therapeutic agents that control cAMP and cGMP levels have been used to treat various cardiovascular diseases.
• 4 The aim of the present review is to discuss: (i) the functions of endothelial cells; (ii) the importance of [Ca²⁺]_i in endothelial cells; (iii) the impact of excessive [Ca²⁺]_i in endothelial cells; and (iv) the balanced control of [Ca²⁺]_i in endothelial cells via involvement of cyclic nucleotides (cAMP and cGMP) and their general effectors.

     

G02 AND Gi3 PROTEINS MEDIATE THE ACTION OF SOMATOSTATIN ON MEMBRANE Ca2+ AND K+ CURRENTS IN OVINE PITUITARY SOMATOTROPHS

1. Growth hormone (GH) secretion from the anterior pituitary gland is mainly regulated by hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIF). Somatostatin reduces both spontaneous and GHRH-stimulated GH secretion. 2. Exocytosis of GH is mainly determined by the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is regulated by the influx of Ca²⁺ via membrane Ca²⁺ channels. Somatostatin reduces the influx of Ca²⁺ through two separate mechanisms, namely a direct action on Ca²⁺ channels and an indirect action on membrane potentials through the activation of K⁺ channels. 3. In the present experiments, somatotroph-enriched cells were obtained from the ovine pituitary gland by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the effect of SRIF (10 nmol/L) on Ca²⁺ or K⁺ currents. 4. A significant reduction in Ca²⁺ currents and an increase in K⁺ currents was obtained in response to local application of SRIF (10 nmol/L), but vehicle application had no effect. The responses of Ca²⁺ and K⁺ currents to SRIF were reversible after removal of SRIF. 5. Dialysis of GTP-λ-s (200 μmol/L) abolished the recovery phase of K⁺ current response to SRIF after its removal, whereas GDP-β-s (200 μmol/L) totally blocked the response. Pretreatment of the cells with pertussis toxin (100 nmol/L) overnight abolished the Ca²⁺ current response to SRIF. 6. Intracellular dialysis of antibodies to αo, α_{1_3}, ai_1-2 and ai₃summits of the G-proteins into cells via whole-cell patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. 7. Dialysis of anti-ai_1-3 or anti-@aLi₃ antibodies significantly attenuated the increase in the K⁺ current in response to 10 nmol/L SRIF, whereas neither anti-αo nor anti-αi_2 antibodies diminished the effect of SRIF on the K⁺ current. 8. Dialysis of anti-αo antibodies significantly attenuated the reduction in the Ca²⁺ current that was obtained upon application of 10 nmol/L SRIF. Neither anti-αi-2 nor anti-αi3 antibody dialysis diminished the effect of SRIF on the Ca²⁺ current. 9. Dialysis of the ao common antisense oligonucleotides (ASm) but not the αi₃ AS significantly diminished the inhibitory effect of SRIF on the Ca²⁺ current. This effect of ao ASm dialysis occurred at 12 h incubation after dialysis, reaching a maximal level at 48 h and partially recovering at 72 h incubation. Antisense oligonucleotides specific for αo₁ (αo₁ AS) or αo₂(α0₂ AS) were dialysed into somatotrophs and only αo₂ AS significantly attenuated the inhibition of SRIF on the Ca²⁺ current. 10. It is concluded that the G_i3 protein mediates the effect of SRIF on the K⁺ current and that the G02 protein mediates the effect of SRIF on the Ca²⁺ current in primary cultured ovine somatotrophs.     

EFFECTS OF CA2+ ANTAGONISTS ON CARDIOVASCULAR RESPONSES TO POSTERIOR HYPOTHALAMIC STIMULATION IN THE RAT

The effects of methoxyverapamil or hydralazine on pressor responses to posterior hypothalamic stimulation and injected pressor agents were studied in normotensive male Wistar rats. Methoxyverapamil inhibited both phases of pressor responses to hypothalamic stimulation and pressor responses to injected noradrenaline or angiotensin II. Hydralazine inhibited the secondary phase (due to adrenomedullary catecholamine) and not the primary phase (due to increased sympathetic vasomotor activity) of pressor response to hypothalamic stimulation. However, it inhibited the pressor responses to exogenous noradrenaline or angiotensin II. The data indicate that hydralazine is ineffective in inhibiting the pressor response elicited by noradrenaline endogenously released at the sympathetic nerve endings.     

THE EFFECT OF CROMAKALIM ON THE ELECTRICAL PROPERTIES OF AND [86Rb+] EFFLUX FROM NORMAL AND HYPERTROPHIED RAT BLADDER

1. The activity of smooth muscle strips from normal and hypertrophied rat bladders was compared. The hypertrophied bladders were produced by partially obstructing the urethra 8–13 weeks previously. 2. Spontaneous mechanical activity was more frequent and smaller in amplitude in strips from normal than hypertrophied bladders and was less sensitive to cromakalim, being reversibly abolished by cromakalim at 10^?6 mol/L compared with 10^?7 mol/L for hypertrophied bladder. 3. The mean resting membrane potentials of smooth muscle cells from normal and hypertrophied rat bladders were -47.2 and -47.6 mV, respectively. Bursts of spontaneous action potentials, corresponding to the mechanical activity, were seen in some cells. 4. Nifedipine at 10^?6 mol/L had no significant effect on the resting membrane potential. Occasional single spikes occurred with increased duration and the afterhyperpolarization was abolished. Cromakalim at 10^?5 mol/L produced hyperpolarization of 3–9 mV and, in the continued presence of the drug, occasional singe spikes could be recorded from both normal and hypertrophied bladders. 5. Nifedipine at 10^?6 mol/L abolished movement but did not significantly alter [⁸⁶Rb⁺] efflux from strips from either normal or hypertrophied bladders. Addition of cromakalim at 5± 10^?6 or 5 ± 10^?5 mol/L in the presence or absence of nifedipine increased efflux from the normal bladder by 30–40%. In the hypertrophied bladder the efflux increased by about 14% and 28% in the presence of 5 ± 10^?6 and 5 ± 10^?5 mol/L cromakalim, respectively. 6. No significant differences between electrical properties of normal and hypertrophied bladders were therefore found and we are unable to explain why the mechanical activity of hypertrophied rat bladders was more sensitive to cromakalim than normal bladders.     

KATP CHANNEL BLOCKING ACTIONS OF QUATERNARY IONS PLAY NO ROLE IN THEIR ANTIPROLIFERATIVE ACTION ON MOUSE LEUKAEMIA AND RAT VASCULAR SMOOTH MUSCLE CELLS IN VITRO

1. The aim of the present study was to investigate the possibility that, in the two cell lines examined, alterations in cell growth caused by lipophilic quaternary ions may involve KATP channels. We examined the effect of tetraphenylphosphonium (TPP), tetraphenylboron (TPB), rhodamine 123, dequalinium chloride (DECA) and the non-quaternary ion cisplatin on the proliferation of L1210 mouse leukaemia cells and rat smooth muscle cells in vitro. The KATP channel opener levcromakalim (LKM) and the Katp channel antagonist glibenclamide were also tested. 2. From growth-inhibition studies, the rank order of potency (based on pIC₅₀ values) using L1210 leukaemia cells was: DECA (6.61) > cisplatin (6.09) = rhodamine 123 (6.01) > TPP (5.61) > TPB (4.25). Levcromakalim and glibenclamide were found to be inactive at the maximum concentrations used (100 μmol/L). A different rank order of potency was obtained in rat aortic smooth muscle cells: cisplatin (6.33) > DECA (5.67) > TPP (4.96) > rhodamine 123 (4.1). Tetraphenylboron (30μmol/L), LKM (100 μmol/L) and glibenclamide (100 μmol/L) were found to be inactive. 3. When the negatively charged TPB (30 μmol/L) was combined with some of the active agents, the potency of the active agents was increased. Thus, in L1210 cells, rhodamine 123, DECA and TPP were all more potent at inhibiting cell growth in the presence of TPB. Tetraphenylboron had no effect on cisplatin in this cell line. In rat smooth muscle cells, TPB (30 μ mol/L) potentiated the effect of rhodamine 123 but had no effect on the actions of cisplatin, DECA or TPP. 4. In functional studies, rhodamine 123 was a weak antagonist of the vasorelaxant responses to the K_ATP channel opener LKM in the porcine right circumflex artery in vitro. The pK_B value obtained for rhodamine 123 at 100 μmol/L was 4.95. Dequalinium chloride was inactive. 5. We found no correlation between the actions of the compounds tested to antagonize K_ATP channels and their ability to inhibit cell proliferation. In addition, compounds known to regulate K_ATP channel activity failed to influence proliferative rates. These results suggest that K_ATP channels are not involved in the antiproliferative action of TPP and other quaternary ions in the two cell lines studied.     

EFFECTS OF BKCa CHANNELS ON THE REDUCTION OF CYTOSOLIC Ca2+ IN cGMP-INDUCED RELAXATION OF GUINEAPIG TRACHEA

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca²⁺ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca²⁺-activated K⁺ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC₇₀ values for ANP and EC₅₀values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca²⁺channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca²+-activated K⁺ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K⁺, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca²⁺-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca²⁺ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca²⁺ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca²⁺ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca²⁺ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca²⁺ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.