Realist analysis of whether emergency departments with primary care services generate 'provider-induced demand'.

BACKGROUND: It is not known whether emergency departments (EDs) with primary care services influence demand for non-urgent care ('provider-induced demand'). We proposed that distinct primary care services in EDs encourages primary care demand, whereas primary care integrated within EDs may be less likely to cause additional demand. We aimed to explore this and explain contexts (C), mechanisms (M) and outcomes (O) influencing demand. METHODS: We used realist evaluation methodology and observed ED service delivery. Twenty-four patients and 106 staff members (including Clinical Directors and General Practitioners) were interviewed at 13 EDs in England and Wales (240 hours of observations across 30 days). Field notes from observations and interviews were analysed by creating 'CMO' configurations to develop and refine theories relating to drivers of demand. RESULTS: EDs with distinct primary care services were perceived to attract demand for primary care because services were visible, known or enabled direct access to health care services. Other influencing factors included patients' experiences of accessing primary care, community care capacity, service design and population characteristics. CONCLUSIONS: Patient, local-system and wider-system factors can contribute to additional demand at EDs that include primary care services. Our findings can inform service providers and policymakers in developing strategies to limit the effect of potential influences on additional demand when demand exceeds capacity.

Peripheral mechanisms II: the pharmacology of peripherally active antitussive drugs.

Cough is an indispensable defensive reflex. Although generally beneficial, it is also a common symptom of diseases such as asthma, chronic obstructive pulmonary disease, upper respiratory tract infections, idiopathic pulmonary fibrosis and lung cancer. Cough remains a major unmet medical need and although the centrally acting opioids have remained the antitussive of choice for decades, they have many unwanted side effects. However, new research into the behaviour of airway sensory nerves has provided greater insight into the mechanisms of cough and new avenues for the discovery of novel non-opioid antitussive drugs. In this review, the pathophysiological mechanisms of cough and the development of novel antitussive drugs are reviewed.

Enhanced vascular responses to adrenomedullin in mice overexpressing receptor-activity-modifying protein 2.

Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activity-modifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin gene-related peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin II-raised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressure-volume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0+/-1.5 nmol/L) than WT (EC50, 17.9+/-3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM(22-52), such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.

Selective inhibition of thapsigargin-induced contraction and capacitative calcium entry in mouse anococcygeus by trifluoromethylphenylimidazole (TRIM).

This study examined the effects of trifluoromethylphenylimidazole (TRIM) on tone, and calcium entry, in mouse anococcygeus stimulated by either thapsigargin (Tg; 100 nM) which activates capacitative calcium entry (CCE), or high K (60 mM) which activates voltage-operated calcium channels. TRIM (1 - 333 microM) produced concentration-related relaxation of Tg-induced tone (EC(50), 42 microM) but was much less effective against high K. In single smooth muscle cells loaded with FURA-2, TRIM reduced the increase in fluorescence ratio produced by Tg but had no effect on that produced by high K. The relaxations of Tg-induced tone, and reduction in fluorescence ratio, were obtained in the presence of L-N(G)-nitroarginine and were thus independent of nitric oxide synthase inhibition; further, TRIM had no discernible effect on nitrergic responses. TRIM provides a novel drug for the selective inhibition of CCE and a template for the development of more potent inhibitors.

Inhibition of capacitative calcium entry is not obligatory for relaxation of the mouse anococcygeus by the NO/cyclic GMP signalling pathway.

1. The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. 2. In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic field stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 microM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microM). 3. In single smooth muscle cells, loaded with Fura-2, both carbachol and Tg produced sustained elevations in cytoplasmic calcium levels ([Ca2+]i). SNP inhibited the rise in [Ca2+]i produced by carbachol, an effect attenuated by ODQ. In contrast, neither SNP nor 8-Br-cyclic GMP reduced the elevated [Ca2+]i associated with Tg. 4. In beta-escin skinned preparations, NO had no effect on tone induced by calcium (1 microM in the presence of 100 microM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. 5. The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by refilling the internal stores, since reduction in [Ca2+]i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation.

Thapsigargin-induced tone and capacitative calcium influx in mouse anococcygeus smooth muscle cells.

The sarcoplasmic reticulum Ca-ATPase inhibitor thapsigargin (Tg; 0.4-100 nM) produced concentration-related, strong and sustained contractions of the mouse-isolated anococcygeus muscle; these contractions were dependent on extracellular calcium but were only partially reduced (by about 50%) in the presence of verapamil (10 and 100 microM). The verapamil-resistant component of the Tg-induced contraction was relaxed by the general calcium entry blockers SKF96365 (0.4-40 microM) and cadmium (50-300 microM), and by the tyrosine kinase inhibitor genistein (10-180 microM). In single smooth muscle cells loaded with Fura-2, addition of Tg (100 nM) to calcium-free medium produced a small, transient increase in fluorescence; subsequent addition of calcium (2.5 mM) produced a larger and sustained increase which was abolished on return to calcium-free conditions, but was only partially reduced by verapamil (10 microM; by about 30%). Manganese quenching of Fura-2 was enhanced in cells treated with Tg. The verapamil-resistant calcium influx was reduced by SKF96365 (20 microM) and to a lesser extent by genistein (40 microM); cadmium (200 microM) produced an initial decrease in fluorescence followed by a marked increase. These results demonstrate that, in the mouse anococcygeus, Tg can cause sustained contractions and elevations of calcium influx in the presence of verapamil; the time-course, calcium dependence and, although to a lesser extent, pharmacology of these effects generally support the proposal that excitation-contraction coupling in this tonic smooth muscle involves sustained capacitative calcium influx.

Correlation between store-operated cation current and capacitative Ca2+ influx in smooth muscle cells from mouse anococcygeus.

In mouse anococcygeus cells, simultaneous measurements of membrane currents and changes in intracellular Ca2+ were obtained using "perforated-patch" whole-cell recordings and Fura-2 microfluorimetry. Carbachol (50 microM) and cyclopiazonic acid (10 microM) produced a biphasic inward current; a transient Ca2+-dependent chloride current (I(ClCa)), followed by a smaller, sustained current (I(DOC)) This response was mirrored by a biphasic increase in the intracellular Ca2+ concentration. SKF96365 (1-{beta-[3-(4-methoxyphenyl) propoxyl]-4-methoxyphenethyl}-1H-imidazole; 10 microM) and Cd2+ (100 microM) inhibited both I(DOC) and the sustained increase in intracellular Ca2+; La3+ (400 microM) inhibited neither response. The results confirm that the non-selective cation current I(DOC) underlies capacitative Ca2+ influx supporting sustained contractions in this tonic smooth muscle.

Capacitative Ca2+ entry and the regulation of smooth muscle tone.

In many non-excitable cells, activation of phospholipase C-linked receptors results in a biphasic increase in the cytosolic Ca2+ concentration; an initial transient increase, owing to the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR), is followed by a much smaller but sustained elevation, which often involves capacitative Ca2+ entry, where depletion of Ca2+ within the ER signals the opening of store-operated Ca2+ channels in the plasma membrane. However, in excitable cells such as smooth muscle, the role of capacitative Ca2+ entry is less clear and the main Ca2+ entry mechanisms responsible for sustained cellular activation have been considered to be either voltage-operated or receptor-operated Ca2+ channels. Although store-regulated Ca2+ entry was known to occur following agonist activation of smooth muscle, it was believed to be important only for the re-filling of the depleted SR and not as a source of activator Ca2+ for the contractile mechanisms. Here, Alan Gibson, Ian McFadzean, Pat Wallace and Christopher Wayman review recent evidence that capacitative Ca2+ entry might indeed be important for the regulation of smooth muscle tone, and that it might provide an important for pharmacological intervention.

Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells.

The properties of the calcium stores coupled to a depletion-operated cation current (IDOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 microM) activated an initial, large ( approximately 200 pA), transient, calcium-dependent chloride current (IClCa) followed by a smaller ( approximately 10 pA) sustained, non-selective cation current (IDOC). Intracellular application of heparin (5 mg ml-1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 microM) activated IDOC but not IClCa; ryanodine (30 microM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of IClCa by carbachol. In the presence of 30 microM, but not 3 microM, ryanodine, carbachol was able to activate IDOC. Cyclopiazonic acid (CPA; 10 microM) abolished the response to carbachol; however, caffeine was still able to activate IClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 microM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4, 5-trisphosphate-(IP3-) sensitive or ryanodine-sensitive calcium stores is able to activate IDOC, and, by extension, capacitative calcium entry in this tissue.

Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus.

1. At a holding potential of -40 mV, carbachol (50 microM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (ICl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I(DOC)). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I(oscil)) superimposed on I(DOC). 2. Carbachol-induced I(oscil) (amplitude 97 +/- 11 pA; frequency 0.26 +/- 0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution. 3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I(oscil) could be restored by re-admission of calcium. The frequency, but not the amplitude, of I(oscil) increased with increasing concentrations of extracellular calcium (0.5-10 mM). 4. Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml(-1) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 microM), prevented the activation of I(oscil) by carbachol. Caffeine (10 mM) activated both ICl(Ca) and I(DOC) and prevented the induction of I(oscil) by carbachol. Caffeine and CPA also abolished I(oscil) in the presence of carbachol, as did both a low (3 microM) and a high (30 microM) concentration of ryanodine. 5. Carbachol-induced I(oscil) was abolished by the general calcium entry blocker SKF 96365 (10 MM) and by Cd2+ (100 microM), but was unaffected by La3+ (400 microM). As found previously, I(DOC) was also blocked by SKF 96365 and Cd2+, but not La3+; the inhibition of I(DOC) preceded the abolition of I(oscil) by 27 s with SKF 96365 and by 30 s with Cd2+. Nifedipine (1 microM) produced a partial inhibition of the carbachol-induced I(oscil) frequency at holding potentials of -20 mV and -60 mV and, in addition, reduced I(DOC) at -60 mV by 18%. 6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I(DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.

Inhibition by sodium nitroprusside of a calcium store depletion-activated non-selective cation current in smooth muscle cells of the mouse anococcygeus.

1. The effects of sodium nitroprusside (SNP) on the non-selective cation current activated in response to intracellular calcium store depletion were studied using the whole-cell patch-clamp technique in single smooth muscle cells isolated from the mouse anococcygeus. Voltage-dependent calcium currents were blocked with extracellular nifedipine, and caesium and tetraethylammonium chloride were used to block voltage-dependent potassium currents. Calcium stores were depleted with caffeine (10 mM), carbachol (50 microM) or cyclopiazonic acid (CPA 10 microM; an inhibitor of the sarcoplasmic reticulum [SR] calcium-ATPase). 2. At a holding potential of -40 mV, both CPA and caffeine activated inward currents which consisted of two clearly distinguishable components; an initial transient current followed by a smaller sustained current. In the case of CPA, the amplitudes of the transient and sustained components were 19.7 +/- 2.1 pA and 3.5 +/- 0.3 pA respectively, whilst the equivalent values for caffeine were 188 +/- 21 and 4.8 +/- 0.3 pA. As described previously, the transient current results from activation of a calcium-dependent chloride conductance whilst the sustained current is a non-selective cation current, activated following intracellular calcium store depletion. 3. The muscarinic receptor agonist, carbachol, also activated a transient followed by a sustained current with amplitudes of 238 +/- 55 and 4.7 +/- 0.5 pA respectively. Superimposed on the sustained current were regular, oscillations of calcium-activated chloride current. 4. Both the transient and the sustained currents activated by CPA were absent in cells pretreated with SNP (10 microM). Application of SNP to a cell following activation of the sustained current by CPA inhibited the current by 88.6 +/- 3.8%. SNP (10 microM) did not inhibit the transient current activated by caffeine but abolished the sustained current. 5. SNP (10 microM) had no effect on the initial transient current activated by carbachol (50 microM). However, it did inhibit the oscillations in the inward current. In recordings from cells bathed in extracellular solution containing the chloride channel blocker, anthracene-9-carboxylic acid (A-9-C; 1 mM), carbachol activated only a sustained current. This current was inhibited by 88.1 +/- 6.5% by a concomitant application of SNP (10 microM) and was absent in cells pretreated with the nitrovasodilator. 6. The effects of SNP on the currents activated by caffeine (10 mM) were mimicked by 8-bromo-cyclic GMP (200 microM); thus the nucleotide had no effect on the transient current activated by caffeine but abolished the sustained current. The effects of SNP, but not those of 8-bromo-cyclic GMP, were inhibited by the nitric oxide-sensitive guanylyl cyclase inhibitor, 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxaline-1-one (ODQ; 1 microM). ODQ alone produced a significant increase in the size of the sustained current activated by caffeine (7.8 +/- 0.7 pA). 7. These findings suggest that SNP activates guanylyl cyclase to inhibit the non-selective cation current activated as a result of intracellular calcium store depletion in mouse anococcygeus cells. Since the non-selective cation current appears to underlie the calcium entry process responsible for maintaining the sustained contractions to agonists in this tissue, this action of SNP may represent an important mechanism by which nitrates relax non-vascular smooth muscle.

Two distinct membrane currents activated by cyclopiazonic acid-induced calcium store depletion in single smooth muscle cells of the mouse anococcygeus.

1. By use of the whole-cell configuration of the patch-clamp technique, membrane currents induced by cyclopiazonic acid (CPA; an inhibitor of the sarcoplasmic reticulum (SR) calcium-ATPase) were investigated in single smooth muscle cells freshly dispersed from the mouse anococcygeus. Voltage-dependent calcium currents were blocked with extracellular nifedipine and caesium and tetraethylammonium chloride were used to block voltage-dependent potassium currents. 2. At a holding potential of -40 mV, CPA (10 microM) activated an inward current that consisted of two distinct components. The first was an initial transient current with an amplitude of 19.6 +/- 1.9 pA while the second was sustained and had an amplitude of 3.5 +/- 0.3 pA. 3. The current-voltage (I-V) relationship for the transient current showed marked outward rectification. The current had a reversal potential of 9.1 +/- 1.1 mV which was shifted to 29.0 +/- 4.2 mV when the extracellular chloride concentration was lowered from 148.4 to 58.4 mM. The sustained current had a near-linear I-V relationship and a reversal potential of 31.0 +/- 2.7 mV. Removal of extracellular calcium had no effect on the transient current, but shifted the reversal potential of the sustained current to 18.2 +/- 5.7 mV. 3. The initial transient current was abolished in cells bathed in extracellular solutions containing the chloride channel blockers, 4,4' diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS; 1 mM) or anthracene-9-carboxylic acid (A-9-C; 1 mM), and was absent in cells containing the calcium buffers EGTA (1 to 5 mM) or BAPTA (10 mM). The second sustained current was unaffected by either the chloride channel blockers or the intracellular calcium buffers. 4. Treatment of the cells with caffeine (10 mM) produced similar inward currents to those produced by CPA. In the presence of caffeine, CPA (10 microM) induced no further inward current. 5. In organ bath studies, CPA (10 microM)-induced contractions of the mouse anococcygeus were inhibited by cadmium and nickel (both 50-400 microM) and the general calcium entry blocker, SKF 96365 (10 microM); lanthanum and gadolinium had no effect at concentrations up to 400 microM. The pharmacology of the CPA-induced non-selective cation current mirrored that of the CPA-induced whole muscle contraction being reversed by cadmium (100 microM) and SKF 96365 (10 microM), but unaffected by lanthanum (400 microM). The initial chloride conductance was unaffected by cadmium, SKF 96365 or lanthanum. 6. It is concluded that CPA activates a transient calcium-dependent chloride current as a consequence of calcium release from intracellular stores; this current would result in depolarization and opening of voltage-operated calcium channels, which mediate the nifedipine-sensitive component of muscle contraction. In addition, as a result of emptying the SR, CPA activates a non-selective cation conductance which may underlie the nifedipine-insensitive calcium entry process utilised during sustained contraction.

Inhibition of voltage-dependent Ca(2+)-current by alpha-adrenoceptor agonists in smooth muscle cells.

The cellular mechanisms underlying the inhibitory effects of phenylephrine on dihydropyridine-sensitive, voltage-dependent Ca2+ currents recorded from single smooth muscle cells dissociated from the rat anococcygeus muscle were examined. Phenylephrine (0.1-30 microM) produced a concentration-dependent inhibition of the Ca2+ current; the maximum response occurred at a concentration of 10 microM, which inhibited the peak inward current evoked at 0 mV by 57.7 +/- 4% (n = 8). The response to phenylephrine was reduced but not abolished in cells containing 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA; 10 mM), and it persisted in cells dialysed internally with heparin (5 mg.ml-1). This was despite the fact that both EGTA (5 mM) and heparin were able to block the phenylephrine-induced, Ca(2+)-dependent chloride current recorded in the same cells. The inhibition of the Ca2+ current produced by phenylephrine was abolished in cells containing guanosine 5'-[beta-thio]diphosphate (GDP-beta-S) but persisted in cells pre-treated with pertussis toxin. Our results suggest that the inhibition of L-type Ca2+ current seen following alpha-adrenoceptor activation occurs by a mechanism independent from the inositol trisphosphate-mediated release of Ca2+ from intracellular stores.

The nitrergic transmitter of the anococcygeus--NO or not?

Nonadrenergic noncholinergic (NANC) relaxations of the anococcygeus muscle are reduced by inhibitors of nitric oxide synthase (NOS). Since NOS can be detected within 6-hydroxydodpamine-resistant nerve tracts running through the muscle, it seems clear that these NANC relaxations result from activation of the L-arginine/NO pathway within the prejunctional nerve terminal, an example of so-called "nitrergic" transmission. However, a number of substances (hydroquinone, superoxide anions, hydroxocobalamin) profoundly reduce relaxations to exogenous NO but do not affect those to nitrergic field stimulation; such observations have raised questions over the nature of the substance actually released from the nitrergic nerves. Several possible explanations are discussed: (1) NO is released attached to a carrier molecule, perhaps in the form of a nitrosothiol; (2) NO is released in a modified redox form; (3) NO is released as a free radical, but is protected within the neuroeffector junction by other substances which preferentially interact with scavenger molecules; and (4) NO is released as a free radical and, because of a rapid and unhindered rate of diffusion over short distances (100-200 microM), it is less susceptible than exogenous NO to scavenger molecules. As yet, there is insufficient experimental evidence to decide which, if any, of these explanations is correct.

Variable potency of nitrergic-nitrovasodilator relaxations of the mouse anococcygeus against different forms of induced tone.

1. U46619 (thromboxane A2 receptors; 0.002-1 microM), carbachol (muscarinic M3 receptors; 0.1-100 microM), cyclopiazonic acid (CPA; Ca(2+)-ATPase inhibitor; 0.1-30 microM) and K+ (5-100 mM) produced concentration-dependent contractions of the mouse isolated anococcygeus muscle. Equi-effective, submaximal concentrations of each agent were used in further experiments (40 nM U46619; 5 microM carbachol; 5 microM CPA; 70 mM K+). 2. Nifedipine (1 microM) totally abolished contractile responses to K+; those to U46619, carbachol and CPA were reduced by only 20-30% in the presence of nifedipine, but were greatly reduced (> 90%) by a combination of nifedipine and SKF 96365 (0.1-40 microM). 3. In Ca(2+)-free medium, contractions to K+ and CPA were abolished. Small residual responses remained to both carbachol and U46619; those to carbachol were transient, could not be repeated in the continued absence of Ca2+ and were prevented by pre-incubation with CPA, but unaffected by SKF 96365; those to U46619 were sustained, could be repeated in the absence of Ca2+, and were resistant to CPA and SKF 96365. 4. Tone induced by all four agents could be relaxed by sodium nitroprusside (SNP), but with a clear order of potency. SNP (pIC40) was most effective against U46619 (7.92), less so against carbachol (6.80) and CPA (6.68), and least potent against K+ (5.94). A similar order of potency was observed with 8Br-cyclic GMP (50 microM) and nitrergic field stimulation (1-20 Hz). 5. The relaxant potency of SNP was similar in normal Krebs solution and in high K+ (70 mM) Krebs containing 1 microM nifedipine. 6. Inclusion of SNP (0.01-1 microM) or 8Br-cyclic GMP (50 microM) in the Ca2+-free medium inhibited the transient residual response to carbachol. Inclusion of similar concentrations of SNP or 8Br-cyclic GMP,during Ca2+ re-loading, increased the subsequent residual contraction to carbachol in Ca2+-free medium.7. At higher concentrations, SNP (0.1-10 microM) produced a partial relaxation of the sustained contraction to U46619 in Ca2+-free medium.8. Thus, the relaxant potency of the nitrergic stimuli was dependent on the agent and mechanism used to induce tone in the preparation. Examination of the contractile/relaxant interactions suggests that altered Ca2+ sequestration and inhibition of contractile protein function may underlie nitrergic relaxations of this tissue.

Nitrergic stimulation does not inhibit carbachol-induced inositol phosphate generation in the rat anococcygeus.

Carbachol (50 microM) produced a rapid, transient increase in inositol-1,4,5-trisphosphate (IP3) levels in the rat anococcygeus; the peak increase observed at 10 s (3-fold above controls) was greatly reduced in the presence of atropine (100 nM), but was unaffected by nitrergic stimulation (10 Hz), sodium nitroprusside (10 microM) or 8-Br-cyclic GMP (200 microM). Following loading of muscles with [3H]myo-inositol, subsequent exposure to carbachol for 30 min resulted in a 6-fold increase in the accumulation of [3H]inositol-1-monophosphate; again, this action of carbachol was greatly attenuated by atropine, but unaffected by nitrergic stimulation, sodium nitroprusside or 8-Br-cyclic GMP. It is concluded that inhibition of agonist-induced generation of inositol phosphates cannot explain the ability of nitrergic activation to relax (by 54-62%) carbachol-induced tone in this tissue.

A chloroform extract of the herb feverfew blocks voltage-dependent potassium currents recorded from single smooth muscle cells.

We have studied the effects of a chloroform extract of fresh leaves from the herb feverfew (Tanacetum parthenium) on potassium currents in smooth muscle. The currents were recorded from single cells dissociated from the rat anococcygeus and the rabbit ear artery using the whole-cell patch-clamp technique. When applied to cells isolated from the rat anococcygeus, the extract reduced the inactivating voltage-dependent potassium current in a concentration-related manner, with an IC50 value (the concentration that reduced the current by 50%) of 56 micrograms mL-1. Complete block of the current occurred at 1 mg mL-1. In addition to reducing the peak current, feverfew decreased the time to peak of the current and increased the rate of decay of the current. These effects can be explained by the feverfew extract blocking open potassium channels. In single cells isolated from rabbit ear artery the feverfew extract again reduced the voltage-dependent potassium current, whilst at the same time having no effect on the spontaneous transient outward currents which arise as a consequence of activation of calcium-dependent potassium channels. These results suggest that chloroform extracts of feverfew leaf contain an as yet unidentified substance capable of producing a selective, open-channel block of voltage-dependent potassium channels.

Properties of the inactivating outward current in single smooth muscle cells isolated from the rat anococcygeus.

The properties of the voltage- and time-dependent outward current in single smooth muscle cells isolated from the rat anococcygeus were studied. The outward current was activated by depolarizations to membrane potentials positive to -40 mV. Activation followed third order kinetics; at +20 mV, the time for the current to reach half its maximal amplitude was around 55 ms. The current inactivated with a time course that could best be described by a single exponential with a time constant around 1500 ms. The steady-state inactivation curve was voltage dependent over the range -110 to -30 mV, with a half-inactivation point of -67 mV. Recovery from inactivation followed an exponential time course with a time constant of around 770 ms at -90 mV. Deactivating tail current analysis revealed that a 10-fold change in the extracellular potassium ion concentration resulted in a 42 mV change in the reversal potential of the current. The current was blocked by 4-aminopyridine, tetraethylammonium, quinine and verapamil with IC50's--the concentrations producing 50% inhibition of the peak current--of 2 mM, 4 mM, 12 microM and 20 microM respectively. The current was not blocked by Toxin I (100 nM) or glibenclamide (10 microM). The current was still present in cells containing 5 mM EGTA; in these cells, replacing extracellular calcium with cadmium depressed the peak current by around 12%. This could be explained, at least in part, by a negative shift in the voltage dependence of inactivation.

Effects of bradykinin on ion conductances in NG108-15 neuroblastoma x glioma hybrid cells recorded with patch-clamp electrodes.

Under whole-cell recording, bradykinin (BK) produced an initial outward membrane current followed by an inward current in voltage-clamped NG108-15 cells. The initial outward current was associated with a rise in intracellular Ca2+ and was accompanied by the opening of Ca(2+)-dependent K(+)-channels recorded with a cell-attached patch electrode. This current was inhibited by intracellular Mg2+. The inward current was associated with inhibition of the voltage-dependent K(+)-current IK(M). These effects accord with those previously observed in microelectrode-impaled cells, with the difference that BK produced much more pronounced and long-lasting desensitization in the patch-clamped cells.