Biomedical subjects

T J Shuttleworth

Publications and source records attributed to T J Shuttleworth.

At least 37 records · Page 2Linked to original sources

PubMed1994

Ca2+ influx drives agonist-activated [Ca2+]i oscillations in an exocrine cell.

In current models describing agonist-induced oscillations in [Ca2+]i, Ca2+ entry is generally assumed to have a simple sustaining role, replenishing Ca2+ lost from the cell and recharging intracellular Ca2+ stores. In cells from the avian nasal gland, a model exocrine cell, we show that inhibition of Ca2+ entry by La3+, SK&F 96365, or by membrane depolarization, rapidly blocks [Ca2+]i oscillations but does so without detectable depletion of agonist-sensitive Ca2+ stores. As the rate of Mn2+ quenching during [Ca2+]i oscillations is constant, Ca2+ entry is not directly contributing to the [Ca2+]i changes and, instead, appears to be involved in inducing the repetitive release of Ca2+ from internal stores. Together, these data contradict current models in that (i) at the low agonist concentrations where [Ca2+]i oscillations are seen, generated levels of Ins(1,4,5)P3 are themselves inadequate to result in a regenerative [Ca2+]i signal, and (ii) Ca2+ entry is necessary to actually drive the intrinsic oscillatory mechanism.

Animals↗

PubMed1994

Vasoactive intestinal peptide stimulates a cAMP-mediated Cl- current in avian salt gland cells.

VIP plays an integral role in both protein and fluid secretion in many exocrine glands. By employing the perforated patch-clamp whole-cell recording technique we investigated the effects of VIP on membrane potential and transmembrane currents in avian exocrine salt gland cells. Prior to application of VIP, salt gland cells had a resting membrane potential close to -45 mV. When challenged with VIP (1-100 nM) a sustained depolarization to ECl- was induced which was mimicked by the application of cell-permeable cAMP analogues or forskolin (1 microM). By employing the voltage-clamp recording configuration a sustained increase in current was observed with a reversal potential which approximated ECl-. Ionic substitution experiments confirmed that the current was a Cl- conductance which was inhibited by the Cl- channel blockers flufenamic acid and niflumic acid and by the inhibitory cAMP isomer, adenosine-3',5'-cyclic monophosphothioate, Rp-isomer. Based on this, and the fact that the kinetic properties of the Cl- current activated by VIP are similar to those activated by cAMP, we propose that VIP-receptor interaction results in the activation of a cAMP-dependent Cl- current.

Animals↗

PubMed1994

Muscarinic-receptor activation stimulates oscillations in K+ and Cl- currents which are acutely dependent on extracellular Ca2+ in avian salt gland cells.

By utilizing the perforated-patch variant of the whole-cell patch-clamp recording technique, in order to maintain the integrity of the normal cellular buffering systems, we demonstrate that carbachol (CCh) stimulates simultaneous oscillations in a Ca(2+)- and voltage-activated K+ current and a linear Ca(2+)-activated Cl- current in an exocrine avian salt gland cell preparation. Similar conductance changes, although sustained rather than oscillatory, are stimulated by the Ca2+ ionophore A23187. The outward K+ current can be inhibited by tetraethylammonium chloride (TEA) whereas the Cl- current is inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino) (NPPB) and N-phenylanthranilic acid (DPC). The oscillations in current stimulated by CCh are acutely dependent on extracellular Ca2+ and are not affected by the application of low doses of caffeine. In addition, the application of caffeine at all doses fails to mimic the current transients stimulated by CCh. As both caffeine and A23187 are unable to stimulate oscillations under the perforated-patch conditions we suggest that in avian salt gland cells the primary oscillatory mechanism probably involves a one-pool mechanism of Ca2+ release which is intimately related to the activation of a Ca2+ influx pathway.

Animals↗

PubMed1994

Temporal relationships between Ca2+ store mobilization and Ca2+ entry in an exocrine cell.

Consideration of the principal current models for agonist-induced activation of Ca2+ entry in electrically non-excitable cells suggests that it may be possible to distinguish between them on the basis of predicted differences in the temporal relationship(s) between intracellular Ca2+ release and the activation of Ca2+ entry. Measurements of changes in [Ca2+]i and Mn2+ quench in individual exocrine cells from the avian nasal gland indicate that, whereas Ins(1,4,5)P3-induced release of intracellular Ca2+ occurs within 3-5 s, the increase in Mn2+ quench is delayed by some 20-30 s. Mn2+ quench rate is similarly increased by thapsigargin, and is blocked by SK&F 96365, indicating that the increased Mn2+ quench observed genuinely reflects agonist-enhanced activity of the divalent cation entry pathway normally traversed by Ca2+. Additional experiments indicate that the observed delay is not due to inhibition of this pathway by elevated [Ca2+]i. Furthermore, the delay cannot be explained by the time required for Ins(1,3,4,5)P4 generation, which is essentially maximal within 10 s of agonist addition. It is concluded that the observed delay in the activation of the Ca2+ entry pathway is best explained by 'capacitative' models where increased entry requires the generation, and transmission to the plasma membrane, of an unknown messenger as a direct result of the depletion of intracellular Ca2+ stores.

Animals↗

PubMed1994

Potentiation of Ca(2+)-activated secretory activity by a cAMP-mediated mechanism in avian salt gland cells.

In the avian salt gland carbachol (CCh) evokes oscillations in K+ and Cl- current that are sufficient to fully activate secretory activity. Employing the perforated patch-clamp technique, we demonstrate that beta-adrenergic receptor activation stimulates a sustained adenosine 3',5'-cyclic monophosphate (cAMP)-dependent Cl- current with no increase in K+ current. This evokes only a modest increase in secretory activity. However, application of isoproterenol in the presence of a threshold dose of CCh results in maximal secretory activity. Membrane potential measurements demonstrate that isoproterenol stimulates a sustained membrane depolarization from approximately -45 mV to the Cl- equilibrium potential (ECl), whereas CCh evokes oscillations in membrane potential to levels more negative than ECl, representing a mixture of K+ and Cl- conductances. We conclude that, in agreement with current models of fluid secretion, maximal stimulation can only be achieved with simultaneous activation of both K+ and Cl- currents. Because isoproterenol fails to stimulate a K+ current, Cl- secretion is reduced as the driving force for Cl- secretion is dissipated. However, if a driving force is imposed by increasing K+ channel activity (by coadministering CCh), Cl- efflux is sustained. These results could provide a basis for the marked potentiation of Ca(2+)-mediated secretion by agonists that increase cAMP seen in in vivo studies of salivary glands and other exocrine tissues.

Animals↗

PubMed1994

Muscarinic receptor characterization in differentiating avian exocrine cells.

The type of muscarinic acetylcholine receptor in exocrine cells of the avian nasal gland in the undifferentiated quiescent (naive) stage and in the partly differentiated salt-secreting (stressed) stage was characterized by ligand binding experiments and by probing receptor messenger RNA with oligonucleotide probes specific for the mammalian receptor subtypes. Competition-binding studies using l-quinuclidinyl [phenyl-4-3H]benzilate and a series of other ligands indicated the presence of only one type of receptor in both cell types. Pharmacological characterization of its ligand-binding properties revealed similarities with the mammalian M3 type. However, 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1 - aminium chloride, generally a partial agonist in cells expressing mammalian M1 receptors, released calcium from intracellular stores in naive and stressed cells. To resolve this, we attempted to characterize the salt gland receptor by molecular means. Northern analysis of salt gland mRNA revealed weak signals only with oligonucleotide probes corresponding to the mammalian m1 receptor type. However, at higher stringencies these signals faded, indicating that the salt gland receptor may resemble the mammalian m1 subtype but has probably a considerable degree of sequence divergence. Such divergence may also explain the observed differences in pharmacological behavior between the avian and the mammalian glandular receptors.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗

PubMed1993

A Gq-type G protein couples muscarinic receptors to inositol phosphate and calcium signaling in exocrine cells from the avian salt gland.

Muscarinic acetylcholine receptor (mAChR) activation in isolated cells from the nasal salt gland of the domestic duck (Anas platyrhynchos) results in a rapid increase in the rate of phosphatidylinositol hydrolysis and pronounced intracellular calcium signals. Both responses can be elicited by treating these cells with fluoroaluminate (AlF4-) indicating the involvement of a heterotrimeric G protein in the transmembrane signaling process. To characterize this G protein, electrophoretically separated membrane proteins were blotted onto nitrocellulose filters and probed with peptide-antibodies raised against portions of different alpha-subunits of mammalian G proteins. We could demonstrate the presence of at least four different G proteins in salt gland cell membranes. Two of these proteins (40 and 41 kD) were ADP-ribosylated by pertussis toxin and were recognized by an antiserum against a common sequence in all G protein alpha-subunits. One protein (46 kD) was a cholera toxin-substrate and was recognized by a Gs-specific antiserum; the other (42 kD) was recognized by Gq-specific antisera and was resistant to ADP-ribosylation. Since the initial inositol phosphate production upon receptor activation with carbachol and the resulting calcium signals were not affected by pertussis toxin-pretreatment of salt gland cells, we conclude that muscarinic receptors are coupled to phospholipase C by a Gq-type G protein.

Animals↗

PubMed1992

Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland.

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

Animals↗

PubMed1992

Ca2+ release from inositol trisphosphate-sensitive stores is not modulated by intraluminal [Ca2+].

In a recent model developed to explain the apparent "quantal" nature of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release from specific intracellular stores, it was proposed that Ca2+ release from the stores may itself be modulated by intraluminal levels of Ca2+, possibly via an action at a binding site on the Ins(1,4,5)P3 receptor/Ca2+ channel complex. Essential predictions of this model include a specific effect of intraluminal Ca2+ levels on the sensitivity of Ins(1,4,5)P3-induced Ca2+ release and a non-exponential decay of passive Ca2+ loss from the store following inhibition of the Ca2+ pump on the store. However, in measurements of Ins(1,4,5)P3-induced Ca2+ release and passive Ca2+ loss in permeabilized preparations of a model exocrine cell under conditions of thapsigargin-induced store depletion, we found that neither of these predicted behaviors could be demonstrated.

Animals↗

PubMed1992

Modulation of inositol(1,4,5)trisphosphate-sensitive calcium store content during continuous receptor activation and its effects on calcium entry.

Changes in intracellular Ca2+ concentration ([Ca2+]i) following the activation of muscarinic receptors with carbachol were studied in cells from the exocrine avian nasal gland that had been maintained in culture for 40-48 h. In these cells, the carbachol-induced sustained increase in [Ca2+]i could be further increased by the subsequent addition of thapsigargin. This increase was due to an additional release of intracellular Ca2+ and a corresponding further enhancement of Ca2+ entry. However, thapsigargin-sensitive and Ins(1,4,5)P3-sensitive stores appeared to be coincident and the initial carbachol stimulus was sufficient to completely empty these stores. It was concluded that the subsequent effect of thapsigargin was due to a partial refilling of the Ins(1,4,5)P3-sensitive stores despite the continued presence of agonist, an effect that was not the result of any decline in levels of cellular Ins(1,4,5)P3 or changes in the generation of Ins(1,3,4,5)P4, which were sustained throughout. Possible explanations for this refilling response include compartmentalization of intracellular Ins(1,4,5)P3, or a desensitization of the Ins(1,4,5)P3 receptor/Ca(2+)-release channel. Alternatively, the data are also compatible with a recently proposed kinetic separation of Ca2+ uptake and release sites. An important implication of this particular interpretation of our findings would be an apparent dependence of Ca2+ entry specifically on the status of the Ca(2+)-uptake component of the agonist-sensitive store, rather than the Ca(2+)-release component.

Animals↗

PubMed1992

Changes in pHi associated with activation of ion secretion in avian nasal salt gland cells.

The fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein (BCECF) was used to determine changes in intracellular pH (pHi) associated with activation of secretion in isolated cells from the salt-secreting avian nasal gland. A correction procedure overcoming artifacts due to BCECF leakage is described. Resting pHi averaged 7.15 +/- 0.03 and was unaffected by the nominal removal of medium HCO3- or by the addition of the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was significantly reduced by amiloride (7.07 +/- 0.02). Muscarinic activation of secretion resulted in a rapid intracellular acidification that was compensated by mechanisms which raised pHi to restore approximately resting levels within 5 min. The principal mechanism involved was amiloride-sensitive and independent of any sustained intracellular Ca2+ concentration change. Recovery of pHi was also aided by HCO3(-)-dependent and DIDS-sensitive mechanisms not seen in the resting cell. The direction of the latter was pHi-dependent, with DIDS further decreasing pHi in acidified cells and increasing pHi in alkalinized cells. This suggests that the DIDS-sensitive pathways are activated under conditions where pHi has been shifted away from resting levels in either direction and act primarily to restore resting pHi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes.

Several recent studies of intracellular Ca2+ concentration ([Ca2+]i using fluorescent indicators have involved experiments performed at nonphysiological temperatures, frequently room temperature. In the studies reported here, a reduction in temperature was shown to influence the measurement of [Ca2+]i in two major ways. The first is an effect on the value for the Kd (apparent dissociation constant) used in the calculation of [Ca2+]i. The Kd values for indo-1 and fura-2 were found to change with temperature in a manner analogous to the related compounds [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid and bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. Experiments showed that the use of previously published Kd values (measured at 37 degrees C) in experiments at room temperature results in errors of at least 25% in the calculated [Ca2+]i. The second arises from the differential temperature sensitivities of the physiological processes that make up any particular value of [Ca2+]i. While resting [Ca2+]i was relatively unaffected by reduced temperature (27 degrees C, 13 degrees C), changes in [Ca2+]i following stimulation were profoundly influenced and in a way that was not simply predictable. Analysis of the responses indicated that the observed effects resulted largely from a temperature-dependent reduction in the rate of Ca2+ removal from the cytosol together with an additional component, namely a marked reduction in Ca2+ entry, at the lower temperature (13 degrees C). The net result was observed as changes in the initial rise in [Ca2+]i on stimulation, together with more profound, and complex, effects on the sustained elevation in [Ca2+]i following stimulation. This latter component was markedly increased at 27 degrees C, but was decreased in cells at 13 degrees C.

Animals↗

PubMed1991

Inositol phosphates and [Ca2+]i signals in a differentiating exocrine cell.

Generation of inositol phosphates and changes in intracellular Ca2+ concentration [( Ca2+]i) upon muscarinic receptor activation were studied in isolated cells from the nasal salt gland of Anas platyrhynchos, comparing responses in the poorly differentiated cells from ducks drinking only tap water (naive cells) with the more fully differentiated actively secreting cells from ducks drinking 1% NaCl solution for 48 h before the experiment (stressed cells). On stimulation, naive cells showed a rapid five- to sevenfold increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], accompanied by similar changes in Ins(1,3,4,5)P4, whereas both values increased only twofold in stressed cells. [3H]quinuclidinyl benzilate binding experiments revealed that these differences in inositol phosphate production were correlated with differences in the numbers of muscarinic acetylcholine receptors. Continuous recordings of [Ca2+]i revealed that Ca2+ release from intracellular stores upon stimulation was similar in both cell types, but the sustained [Ca2+]i signal (dependent on Ca2+ entry) was three times more pronounced in stressed cells. The results suggest that the adaptive differentiation of salt gland cells is associated with the increased expression of the receptor-mediated Ca2+ entry mechanism. In addition, the high rate of phosphoinositide hydrolysis in naive cells upon receptor activation may have a significance in cell growth, proliferation, and differentiation, which are elements of the development of the salt transport capabilities in these cells.

Animals↗

PubMed1990

Fluoroaluminate activation of different components of the calcium signal in an exocrine cell.

In isolated cells from the avian supra-orbital nasal gland, used as a model for exocrine ion secretion, addition of NaF (2-15 mM) produced a slow Al3(+)-enhanced increase in intracellular Ca2+ concn. ([Ca2+]i), resulting in a more than 2-fold sustained elevation in [Ca2+]i. Simultaneously, cellular Ins(1,4,5)P3 contents became markedly elevated, suggesting an AlF4- activation of a phospholipase C-specific G-protein. Subsequent addition of the muscarinic agonist carbachol failed to produce any further sustained increase in [Ca2+]i, indicating that the AlF4(-)-induced increase in [Ca2+]i involves a Ca2(+)-entry pathway identical with that activated by carbachol. In low-Ca2+ media (extracellular [Ca2+] = 0.04 mM) no such increase in [Ca2+]i, either sustained or transient, is seen, although cellular Ins(1,4,5)P3 levels were markedly elevated. Despite the failure to observe any change in [Ca2+]i in the low-Ca2+ medium, estimation of the size of the agonist-sensitive Ca2+ stores (determined as the magnitude of the transient change in [Ca2+]i induced by carbachol) revealed that these are progressively emptied by the action of AlF4-. However, the onset of this emptying showed an initial lag period of at least 2 min (with 5 mM-NaF plus 10 microM-AlCl3). In marked contrast, determinations of the magnitude of the Ca2(+)-entry pathway under identical conditions showed that this was significantly activated after as little as 1 min of AlF4- treatment. This suggests that, under these conditions, activation of Ca2+ entry in these cells preceded the release of Ca2+ from agonist-sensitive stores, contradicting current models in which the receptor-enhanced entry of extracellular Ca2+ is entirely dependent on, and subsequent to, the prior release of Ca2+ from the intracellular stores.

Aluminum↗

PubMed1990

Receptor-activated calcium entry in exocrine cells does not occur via agonist-sensitive intracellular pools.

Currently, most models describing receptor-activated Ca2+ entry in exocrine cells invoke a pathway for the entry of extracellular Ca2+ directly linking the agonist-sensitive intracellular Ca2+ pools with the plasma membrane. In the avian nasal gland, a model exocrine ion-secreting tissue, we have found that Ca2+ entry during refilling of the intracellular pools following termination of receptor activation (by atropine) occurs via the cytoplasm and not directly into the empty pools. Under appropriate conditions this can be demonstrated as a transient increase in [Ca2+]i (intracellular Ca2+ concn.) seen on restoration of normal extracellular Ca2+ concentrations after atropine to stimulated cells whose intracellular stores have been prevented from refilling by incubation in a low-extracellular-Ca2+ medium. The magnitude of these [Ca2+]i transients decays with time, but with a time course markedly slower than for the corresponding decrease in intracellular Ins(1,4,5)P3. Further experiments have revealed that Ca2+ entry into the cytoplasm during the initial stimulation phase is also direct and not via the intracellular pools. Thus the initial rates of increase in [Ca2+]i during stimulation are always faster in conditions where both Ca2+ entry and Ca2+ release occur (i.e. they are additive). These differences could not be explained by any effects of extracellular Ca2+ on the initial increases in intracellular Ins(1,4,5)P3 after addition of carbachol. These data are therefore inconsistent with the current models in which the rate of Ca2+ entry through the agonist-sensitive pools cannot exceed the rate of Ca2+ release. It appears therefore that Ca2+ entry and Ca2+ release must occur via separate pathways operating in parallel, and not in series as previously predicted.

Animals↗

PubMed1989

Intracellular [Ca2+] and inositol phosphates in avian nasal gland cells.

Isolated cells from the nasal salt gland of ducklings (Anas platyrhynchos) were evaluated as a model system for the study of the muscarinic activation of exocrine ion secretion. Cells loaded with the fluorescent probe indo-1 were used to study changes in intracellular Ca2+ concentration [( Ca2+]i) after stimulation. Changes in inositol phosphate generation and oxygen consumption were also determined. Loading with the acetomethoxy ester form of indo-1 (indo-1/AM) was rapid, and intracellular cleavage of the ester was essentially complete. Leakage of the dye was negligible over the time course of measurements (up to 20 min). Resting [Ca2+]i was approximately 100 nM. Stimulation with carbachol resulted in progressive increases in the generation of inositol phosphates and rapid four- to fivefold increases in [Ca2+]i. At normal extracellular Ca2+ concentrations, [Ca2+]i remained elevated (approximately 3 times resting levels) for as long as stimulation continued. Experiments showed that the increases in [Ca2+]i were comprised of a combination of release of Ca2+ from intracellular stores and an enhanced entry of Ca2+ from the extracellular medium. It is specifically this latter process that produces the sustained elevations in [Ca2+]i that are the essential signal for secretory activity.

Animals↗

PubMed1987

A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors.

A peptide that cross reacted with N-terminal, but not C-terminal, antisera to vasoactive intestinal peptide (VIP) was isolated from extracts of intestine from the dogfish Scyliorhinus canicula. Microsequence analysis gave the structure His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ile-Arg-Lys-Gln-Met-Ala-Val-Lys - Lys-Tyr-Ile-Asn-Ser-Leu-Leu-Ala-NH2. C-terminal amidation was determined by HPLC analysis of phenylthiocarbamyl amino acid derivatives after carboxypeptidase Y digestion. The peptide differs at five positions from the porcine octacosapeptide. Dogfish VIP was equipotent with its porcine counterpart in inhibiting binding of 125I-labelled VIP to guinea pig dispersed pancreatic acini, and in stimulating amylase secretion by the same preparation. The data indicate a strong conservation of VIP during evolution and permit identification of residues crucial for bioactivity.

Amino Acid Sequence↗

PubMed1987

Sites of interactions of surfactants with beta-adrenergic responses in trout (Salmo gairdneri) gills.

The aims of this study were to investigate the effects of surfactants on the beta-adrenergic responses of the arterio-arterial (lamellar) vascular pathway of isolated gills from the trout Salmo gairdneri. The resistance of the arterio-arterial vasculature was monitored by measuring the input pressure of isolated gills perfused at a constant flow and at constant efferent pressure. Specific beta-adrenergic stimulation with isoprenaline produced a dose-dependent reduction in vascular resistance which was mimicked by the presence of cyclic 8-(4-chlorophenylthio) adenosine-3':5'-monophosphate (8-cpt cyclic AMP). These results suggest that cyclic AMP is involved as an intracellular second messenger of beta-adrenergic regulatory responses of branchial haemodynamics. Surfactants in the perfusate at nominal concentrations of 1.0 mumol 1(-1) (0.3-0.5 mg 1(-1)) inhibited the response of the branchial tissue to isoprenaline but did not affect the response to 8-cpt cyclic AMP. This suggests that the surfactant effects are the result of events occurring within the cell membrane. Furthermore because the surfactants used affected the efficacy, rather than the affinity, of the isoprenaline response then the mechanism of action is unlikely to be an effect on agonist/receptor interaction but may be further along the stimulus response chain, perhaps affecting receptor/regulatory nucleotide/adenylate cyclase interaction or via direct inhibition of the cyclase itself.

Animals↗