Novel alternatively spliced exon in the extracellular ligand-binding domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) selectively increases ligand affinity and alters signal transduction coupling during spermatogenesis.

The expression of the paracrine signaling hormone pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated in a cyclical fashion during the 12-day spermatogenic cycle of the adult rat testis. The precise functions of PACAP in the development of germ cells are uncertain, but cycle- and stage-specific expression may augment cAMP-regulated gene expression in germ cells and associated Sertoli cells. Here we report the existence of a heretofore unrecognized exon in the extracellular domain of the PACAP type 1 receptor (PAC1R) that is alternatively spliced during the spermatogenic cycle in the rat testis. This splice variant encodes a full-length receptor with the insertion of an additional 72 base pairs encoding 24 amino acids (exon 3a) between coding exons 3 and 4. The PAC1R(3a) mRNA is preferentially detected in seminiferous tubules and is expressed at the highest levels in round spermatids and Sertoli cells. Analyses of ligand binding and signaling functions in stably transfected HEK293 cells expressing the two receptor isoforms reveals a 6-fold increase in the affinity of the PAC1R(3a) to bind PACAP-38, and alterations in its coupling to both cAMP and inositol phosphate signaling pathways relative to the wild type PAC1R. These findings suggest that the extracellular region between coding exons 3 and 6 of PAC1R may play an important role in the regulation of the relative ligand affinities and the relative coupling to G(s) (cAMP) and G(q) (inositol phosphates) signal transduction pathways during spermatogenesis.

Regulated expression of adenosine triphosphate-sensitive potassium channel subunits in pancreatic beta-cells.

The regulation of glucose-dependent insulin secretion in pancreatic beta-cells is linked to the expression and function of the ATP-sensitive potassium channel (K(ATP)), which is composed of a sulfonylurea receptor (SUR1) and an inwardly rectifying potassium channel (Kir6.2). Previous animal and human genetic studies have demonstrated that disruption or defective expression of K(ATP) subunit genes has a profound impact on the regulation of insulin secretion. Little is known about how SUR1 and Kir6.2 gene expression is regulated. Here we show that high glucose concentrations lead to a marked decrease (approximately 70%) in Kir6.2 messenger RNA (mRNA) levels in isolated rat pancreatic islets as well as in the INS-1 beta-cell line. This effect is reversible, because exposure to low glucose reinduces Kir6.2 transcript levels. The cognate K(ATP) channel subunit SUR1 showed similar down-regulation at high glucose concentration. The K(ATP) channel activity of INS-1 cells cultivated at high glucose was reduced by 33-51%. In contrast, glucagon-like peptide-1 (GLP-1) induced Kir6.2 mRNA steady state levels and was able to prevent glucose-dependent inhibition of Kir6.2 mRNA and K(ATP) channel activity. To provide further insight into the mechanisms by which glucose and GLP-1 regulate beta-cell K(ATP) channel genes, we have cloned and initiated the characterization of the Kir6.2 gene transcriptional regulatory regions contained within the entire 4.5 kb flanked by the SUR1 and Kir6.2 genes. Transient transfection experiments with five deletion constructs in a pancreatic beta-cell line (INS-1) showed that the proximal 988 bp of the Kir6.2 promoter sequence contributes only 25-30% to the total basal promoter activity. The minimal promoter region -67/+140, also encompassing parts of the 5'-untranslated region, confers sensitivity to GLP-1, which stimulates transcriptional activity of the Kir6.2 minigene by about 2-fold. We propose that glucose- and GLP-1-dependent regulation of K(ATP) subunit genes may be important in the adaptation of beta-cells to changes in secretory demands in physiological and diseased states.

Expression of cAMP-regulated guanine nucleotide exchange factors in pancreatic beta-cells.

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) binds to a Gs-coupled receptor on pancreatic beta-cells and potentiates glucose-induced insulin secretion, insulin gene transcription, and beta-cell growth. These stimulatory effects have been attributed to the elevation of intracellular cAMP levels, though it is now apparent that some stimulatory effects of GLP-1 occur independently of the cAMP-mediated activation of protein kinase A (PKA). The nature of this alternative, PKA-independent signaling pathway remains unknown. Here we present evidence for the expression of type 1 and type 2 cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in beta-cells. GEFs are activated by their binding of cAMP. Because cAMP-GEFs activate Ras/MAPK proliferation signaling pathways, they may play an important role in PKA-independent, GLP-1-mediated, signaling pathways in the regulation of beta-cell growth and differentiation.

Insulinotropic toxins as molecular probes for analysis of glucagon-likepeptide-1 receptor-mediated signal transduction in pancreatic beta-cells.

Cholera toxin, pertussis toxin, mastoparan, maitotoxin, and alpha-latrotoxin are complex protein or polyether-based toxins of bacterial, insect, or phytoplankton origin that act with high potency at the endocrine pancreas to stimulate secretion of insulin from beta-cells located in the islets of Langerhans. The remarkable insulinotropic properties of these toxins have attracted considerable attention by virtue of their use as selective molecular probes for analyses of beta-cell stimulus-secretion coupling. Targets of the toxins include heptahelical cell surface receptors, GTP-binding proteins, ion channels, Ca(2+) stores, and the exocytotic secretory apparatus. Here we review the value of insulinotropic toxins from the perspective of their established use in the study of signal transduction pathways activated by the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). Our analysis of one insulinotropic toxin (alpha-latrotoxin) leads us to conclude that there exists a process of molecular mimicry whereby the 'lock and key'analogy inherent to hormone-receptor interactions is reproduced by a toxin related in structure to GLP-1.

cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic beta-cells. A Ca2+ signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7-37).

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes mellitus. In vitro studies of pancreatic islets of Langerhans demonstrated that GLP-1 interacts with specific beta-cell G protein-coupled receptors, thereby facilitating insulin exocytosis by raising intracellular levels of cAMP and Ca2+. Here we report that the stimulatory influence of GLP-1 on Ca2+ signaling results, in part, from cAMP-dependent mobilization of ryanodine-sensitive Ca2+ stores. Studies of human, rat, and mouse beta-cells demonstrate that the binding of a fluorescent derivative of ryanodine (BODIPY FL-X ryanodine) to its receptors is specific, reversible, and of high affinity. Rat islets and BTC3 insulinoma cells are shown by reverse transcriptase polymerase chain reaction analyses to express mRNA corresponding to the type 2 isoform of ryanodine receptor-intracellular Ca2+ release channel (RYR2). Single-cell measurements of [Ca2+]i using primary cultures of rat and human beta-cells indicate that GLP-1 facilitates Ca2+-induced Ca2+ release (CICR), whereby mobilization of Ca2+ stores is triggered by influx of Ca2+ through L-type Ca2+ channels. In these cells, GLP-1 is shown to interact with metabolism of D-glucose to produce a fast transient increase of [Ca2+]i. This effect is reproduced by 8-Br-cAMP, but is blocked by a GLP-1 receptor antagonist (exendin-(9-39)), a cAMP antagonist ((Rp)-cAMPS), an L-type Ca2+ channel antagonist (nimodipine), an antagonist of the sarco(endo)plasmic reticulum Ca2+ ATPase (thapsigargin), or by ryanodine. Characterization of the CICR mechanism by voltage clamp analysis also demonstrates a stimulation of Ca2+ release by caffeine. These findings provide new support for a model of beta-cell signal transduction whereby GLP-1 promotes CICR by sensitizing intracellular Ca2+ release channels to the stimulatory influence of cytosolic Ca2+.

Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus.

Previously we demonstrated the expression of the long form of the leptin receptor in rodent pancreatic beta-cells and an inhibition of insulin secretion by leptin via activation of ATP-sensitive potassium channels. Here we examine pancreatic islets isolated from pancreata of human donors for their responses to leptin. The presence of leptin receptors on islet beta-cells was demonstrated by double fluorescence confocal microscopy after binding of a fluorescent derivative of human leptin (Cy3-leptin). Leptin (6.25 nM) suppressed insulin secretion of normal islets by 20% at 5.6 mM glucose. Intracellular calcium responses to 16.7 mM glucose were rapidly reduced by leptin. Proinsulin messenger ribonucleic acid expression in islets was inhibited by leptin at 11.1 mM, but not at 5.6 mM glucose. Leptin also reduced proinsulin messenger ribonucleic acid levels that were increased in islets by treatment with 10 nM glucagon-like peptide-1 in the presence of either 5.6 or 11.1 mM glucose. These findings demonstrate direct suppressive effects of leptin on insulin-producing beta-cells in human islets at the levels of both stimulus-secretion coupling and gene expression. The findings also further indicate the existence of an adipoinsular axis in humans in which insulin stimulates leptin production in adipocytes and leptin inhibits the production of insulin in beta-cells. We suggest that dysregulation of the adipoinsular axis in obese individuals due to defective leptin reception by beta-cells may result in chronic hyperinsulinemia and may contribute to the pathogenesis of adipogenic diabetes.

Expression of adenylyl cyclase subtypes in pancreatic beta-cells.

Activation of adenylyl cyclase by Gs-coupled receptors for insulinotropic hormones such as glucagon-like peptide-1 and pituitary adenylate cyclase-activating polypeptide plays a critical role in stimulating glucose-induced insulin secretion. Despite this important role of insulinotropic hormones in the regulation of insulin secretion, little is known about which of the multiple subtypes of adenylyl cyclase are expressed in beta-cells. Here we report the use of PCR primers designed to amplify all subtypes of adenylyl cyclase from cDNA prepared from human and rat islets and from insulin-secreting beta-cell lines. PCR products were cloned and sequenced to identify the subtypes of adenylyl cyclase amplified. Adenylyl cyclase types V and VI, known to couple to Galphas and Gbetagamma in the cAMP signaling pathway, account for all subtypes identified in human islets and INS-1 cells and the majority of subtypes in rat islets and HIT-T15 cells. These findings indicate that pancreatic beta-cells are particularly well suited to transmit signals via Gs-coupled receptors such as that for glucagon-like peptide-1.

A role for Ca2+-sensitive nonselective cation channels in regulating the membrane potential of pancreatic beta-cells.

The incretin hormones, glucagon-like peptide 1 and pituitary adenylyl cyclase-activating polypeptide, are proposed to activate a maitotoxin (MTX)-sensitive, Ca2+-dependent nonselective cation current in pancreatic beta-cells and insulinoma cells. This MTX-sensitive current is present in human beta-cells as well as in mouse and rat beta-cells, and is accompanied by a rise in cytosolic Ca2+ in voltage-clamped cells in which the activation of voltage-dependent Ca2+ channels is prevented. Activation of the nonselective cation current is inhibited by reduction of disulfide bonds with intracellular, but not extracellular, dithiothreitol, and is also abolished by intracellular dialysis with trypsin. The nonselective cation channels that carry this current have a conductance of about 30 pS, with Na+ as the major extracellular cation. We estimate that these cation channels are expressed on beta-cells at a density similar to that of ATP-sensitive potassium channels (K(ATP) channels) and exhibit spontaneous activity at basal glucose concentrations. We propose that this spontaneous cation channel activity constitutes at least part of the depolarizing background conductance that permits changes in the activity of K(ATP) channels to regulate the resting potential of beta-cells.

Insulinotropic glucagon-like peptide-1-mediated activation of non-selective cation currents in insulinoma cells is mimicked by maitotoxin.

Maitotoxin (MTX) activates a Ca2+-dependent non-selective cation current (ICa-NS) in insulinoma cells whose time course is identical to non-selective cation currents activated by incretin hormones such as glucagon-like peptide-1 (GLP-1), which stimulate glucose-dependent insulin secretion by activating cAMP signaling pathways. We investigated the mechanism of activation of ICa-NS in insulinoma cells using specific pharmacological reagents, and these studies further support an identity between MTX- and GLP-1-activated currents. ICa-NS is inhibited by extracellular application of genistein, econazole, and SKF 96365. This inhibition by genistein suggests that tyrosine phophorylation may play a role in the activation of ICa-NS. ICa-NS is not inhibited by incubation of cells in glucose-free solution, by extracellular tetrodotoxin, nimodipine, or tetraethylammonium, or by intracellular dialysis with 4-aminopyridine, ATP, ryanodine, or heparin. ICa-NS is also not significantly inhibited by staurosporine, which does, however, partially inhibit the MTX-induced rise of intracellular Ca2+ concentration. These effects of staurosporine suggest that protein kinase C may not be involved in the activation of ICa-NS but that it may regulate intracellular Ca2+ release. Alternatively, ICa-NS may have a small component that is carried through separate divalent cation-selective channels that are inhibited by staurosporine. ICa-NS is neither activated nor inhibited by dialysis with KF, KF + AlF3 or GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), suggesting that GTP-binding proteins do not play a major role in the activation of this current.

Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells.

In the genetic mutant mouse models ob/ob or db/db, leptin deficiency or resistance, respectively, results in severe obesity and the development of a syndrome resembling NIDDM. One of the earliest manifestations in these mutant mice is hyperinsulinemia, suggesting that leptin may normally directly suppress the secretion of insulin. Here, we show that pancreatic islets express a long (signal-transducing) form of leptin-receptor mRNA and that beta-cells bind a fluorescent derivative of leptin (Cy3-leptin). The expression of leptin receptors on insulin-secreting beta-cells was also visualized utilizing antisera generated against an extracellular epitope of the receptor. A functional role for the beta-cell leptin receptor is indicated by our observation that leptin (100 ng/ml) suppressed the secretion of insulin from islets isolated from ob/ob mice. Furthermore, leptin produced a marked lowering of [Ca2+]i in ob/ob beta-cells, which was accompanied by cellular hyperpolarization and increased membrane conductance. Cell-attached patch measurements of ob/ob beta-cells demonstrated that leptin activated ATP-sensitive potassium channels (K(ATP)) by increasing the open channel probability, while exerting no effect on mean open time. These effects were reversed by the sulfonylurea tolbutamide, a specific inhibitor of K(ATP). Taken together, these observations indicate an important physiological role for leptin as an inhibitor of insulin secretion and lead us to propose that the failure of leptin to inhibit insulin secretion from the beta-cells of ob/ob and db/db mice may explain, in part, the development of hyperinsulinemia, insulin resistance, and the progression to NIDDM.

Signal transduction of PACAP and GLP-1 in pancreatic beta cells.

PACAP and GLP-1 depolarize pancreatic beta cells and stimulate insulin secretion in the presence of glucose. Depolarization occurs through at least two distinct mechanisms: (1) closure of ATP-sensitive K+ channels, and (2) activation of nonselective cation channels (NSCCs). Under physiological conditions the NSCCs carry a predominantly Na(+)-dependent current. The current may also have a Ca2+ component, but this remains to be determined. Acting together, these two signaling systems reinforce each other and serve to promote membrane depolarization, a rise of [Ca2+]i, and exocytosis of insulin-containing secretory granules. The NSCCs in beta cells are dually regulated by intracellular cAMP and [Ca2+]i. In view of this dual regulation, it appears likely that NSCC channel activation results from signaling events occurring not only at the plasma membrane (gating of channels by cAMP; protein kinase A-mediated phosphorylation of channels) but also at intracellular sites (mobilization of calcium stores by an as yet to be determined process). It is noteworthy that activation of NSCCs has also been reported following stimulation of beta-cells with maitotoxin, or after depletion of intracellular Ca2+ stores. Therefore, the possibility arises that PACAP, GLP-1, and maitotoxin all act on the same types of ion channels in these cells, and that these channels are sensitive to alterations in the content of intracellular calcium. FIGURE 6 summarizes our current knowledge concerning the properties of the PACAP and GLP-1 signaling systems as they pertain to the regulation of NSCCs and intracellular calcium homeostasis in the beta cell. Given that PACAP and GLP-1 are proven to be exceptionally potent insulin secretagogues, it is of considerable interest to determine their usefulness as blood glucose-lowering agents. Initial evaluations of the therapeutic effectiveness of GLP-1 indicate a role for this peptide in the treatment of NIDDM, and also possibly insulin-dependent diabetes mellitus (IDDM). A very attractive feature of such a strategy is the demonstrated lack of hypoglycemic side effects attendant to administration of GLP-1 to diabetic subjects. These observations reinforce the notion that peptides of the PACAP/glucagon/VIP family represent important pharmacological tools for use in experimental therapeutics.

Activation of a cAMP-regulated Ca(2+)-signaling pathway in pancreatic beta-cells by the insulinotropic hormone glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone that is currently under investigation for use in the treatment of diabetes mellitus. To investigate the Ca2+ signaling pathways by which GLP-1 may stimulate the secretion of insulin from pancreatic beta-cells, we examined its effects on the concentration of free intracellular Ca2+ ([Ca2+]i) while simultaneously determining what action it exerts on ion channel function. Measurements of [Ca2+]i were obtained from single rat beta-cells and from beta TC6 and HIT-T15 insulinoma cells loaded with the Ca2+ indicator fura-2, and changes in membrane potential and current were monitored using the perforated patch clamp technique. We report a previously undocumented action of GLP-1 and analogs of cAMP (8-bromo-cAMP, Sp- or Rp-adenosine 3',5'-cyclic monophosphothionate triethylamine) to raise [Ca2+]i that is attributable to the activation of a prolonged inward current designated here as IcAMP. Activation of IcAMP is associated with an increased membrane conductance, membrane depolarization, and triggers large increases of [Ca2+]i. IcAMP is primarily a Na+ current that is blocked by extracellularly applied La3+ or by intracellular administration of Ca2+ chelators (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl, EGTA) and which exhibits a reversal potential of about -26 mV. We propose that IcAMP results from the opening of nonselective cation channels that are activated by intracellular Ca2+ and cAMP and which might play an important role in the regulation of insulin secretion from pancreatic beta-cells.

Pituitary adenylate cyclase-activating polypeptide induces the voltage-independent activation of inward membrane currents and elevation of intracellular calcium in HIT-T15 insulinoma cells.

The secretion of insulin by pancreatic beta-cells is controlled by synergistic interactions of glucose and hormones of the glucagon-related peptide family, of which pituitary adenylate cyclase-activating polypeptide (PACAP) is a member. Here we show by simultaneous recording of intracellular calcium ion ([Ca2+]i) and membrane potential that both PACAP-27 and PACAP-38 depolarize HIT-T15 cells and raise [Ca2+]i. PACAP stimulation can result in membrane depolarization by two distinct mechanisms: 1) PACAP reduces the membrane conductance and increases membrane excitability; and 2) PACAP activates a pronounced inward current that is predominantly a Na+ current, blockade by La3+, and which exhibits a reversal potential of about -28 mV. Activation of this current does not require membrane depolarization, because the response is observed when cells are held under voltage clamp at -70 mV. This current may result from the cAMP-dependent activation of nonspecific cation channels because the current is also observed in response to forskolin or membrane-permeant analogs of cAMP. We also suggest that PACAP raises [Ca2+]i and stimulates insulin secretion by three distinct mechanisms: 1) depolarization activates Ca2+ influx through L-type voltage-dependent calcium channels, 2) mobilization of intracellular Ca2+ stores, and 3) entry of Ca2+ via voltage-independent Ca2+ channels. These effects of PACAP may play an important role in a neuro-entero-endocrine loop regulating insulin secretion from pancreatic beta-cells during the transition period from fasting to feeding.

Voltage-independent calcium channels mediate slow oscillations of cytosolic calcium that are glucose dependent in pancreatic beta-cells.

Pancreatic beta-cells and HIT-T15 cells exhibit oscillations of cytosolic calcium ([Ca2+]i) that are dependent on glucose metabolism and appear to trigger pulsatile insulin secretion. Significantly, differences in the pattern of this [Ca2+]i oscillatory activity may have important implications for our understanding of how glucose homeostasis is achieved during the feeding and fasting states. When single beta-cells are exposed to a stepwise increase in glucose concentration that mimics the transition from fasting to feeding states, fast irregular oscillations of [Ca2+]i are observed. Alternatively, when single beta-cells are equilibrated in a steady state concentration of glucose that mimics the fasting state, slow periodic oscillations of [Ca2+]i are noted. Here we report a fundamental difference in the mechanism by which glucose induces these two types of [Ca2+]i oscillatory activity. In agreement with previous studies, we substantiate a role for L-type voltage-dependent Ca2+ channels as mediators of the fast oscillations of [Ca2+]i observed after a stepwise increase in glucose concentration. In marked contrast, we report that voltage-independent calcium channels (VICCs) mediate slow oscillations of [Ca2+]i that occur when beta-cells are equilibrated in steady state concentrations of glucose. Slow [Ca2+]i oscillations are mediated by VICCs which are pharmacologically and biophysically distinguishable from voltage-dependent Ca2+ channels that mediate fast oscillations. Specifically, slow [Ca2+]i oscillations are blocked by extracellular La3+, but not by nifedipine, and are independent of changes in membrane potential. Measurement of membrane conductance also indicate an important role for VICCs, as demonstrated by a steady state inward Ca2+ current that is blocked by La3+. The steady state Ca2+ current appears to generate slow [Ca2+]i oscillations by triggering Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, a process that is mimicked by extracellular application of caffeine, a sensitizer of the ryanodine receptor/Ca2+ release channel. Depletion of intracellular Ca2+ stores with thapsigargin stimulated Mn2+ influx, suggesting the presence of Ca(2+)-release-activated Ca2+ channels. Taken together, these observations are consistent with a role for VICCs (possibly G-type channels) and/or Ca(2+)-release-activated Ca2+ channels as mediators of slow [Ca2+]i oscillations in beta-cells. We propose that slow oscillations of [Ca2+]i probably serve as important initiators of insulin secretion under conditions in which tight control of glucose homeostasis is necessary, as is the case during the fasting normoglycemic state.

Acetylcholine receptor/channel molecules of insects.

Acetylcholine-gated ion channels of the nicotinic type are abundant in the nervous system of insects. The channels are permeable to Na+, K+ and probably Ca(2+), and unlike most vertebrate neuronal nicotinic acetylcholine receptors the receptor/channel molecule is blocked by alpha-bungarotoxin (alpha-Bgt). Such alpha-Bgt-sensitive receptors are present at synapses and on cell bodies of insect neurones. Single channel recordings have shown the existence of multiple conductances of nAChRs. Studies on several different insect preparations have provided evidence for more than one open state and several closed states of insect nAChRs. Functional insect nAChR channels have now been investigated in situ, following reconstitution of a purified protein in bilayers, and as a result of expressing in Xenopus oocytes messenger RNA encoding receptor subunits.

Multiple conductances of neuronal nicotinic acetylcholine receptors.

In the presence of acetylcholine, cationic channels with three different conductances were recorded from neurones of the dissociated housefly (Musca domestica). Large conductance (80 pS) channels, resembling those that are abundant in reconstitution studies with a 65 kDa alpha-bungarotoxin affinity purified polypeptide, were detected in situ. The two larger conductance channels (80 pS; 32 pS) exhibited open and closed times that were best fitted by multiple exponential functions, indicating the presence of at least two open states. A third conductance (20 pS) showed brief, sparse openings and was least frequently observed. The 32 pS channel was the most common.

Nitromethylene actions on in situ and expressed insect nicotinic acetylcholine receptors.

Single channel recordings from dissociated housefly (Musca domestica) neurons show that a novel type of nitromethylene insecticide, 2(nitro-methylene)tetrahydro-1,3-thiazine (NMTHT) gates a channel, the conductance and open time histogram of which resemble those obtained when acetylcholine is the agonist. Injection into Xenopus oocytes of a locust (Schistocerca gregaria) alpha-subunit mRNA results in the expression of functional nicotinic receptors sensitive to NMTHT. Control oocytes injected with distilled water are insensitive to the same concentration of this compound. Thus NMTHT exhibits agonist actions at both in situ and expressed insect nicotinic receptors, and one site of action of this compound is on an insect nicotinic receptor alpha-subunit.

Embryonic and adult neurones of the housefly (Musca domestica) in culture.

This paper provides a detailed description of the dissociation and maintenance in culture of cells derived from embryonic tissue and from the central nervous system of adult housefly, Musca domestica. Dissociation of embryonic tissue produced several cell types of which muscle and neurone-like cells predominated. These cells were morphologically distinct once development had started in vitro with muscle cells often producing rhythmic contractions. Neurones had small cell bodies (less than 10 mum) and showed extensive outgrowth of neurites. Neuronal cell bodies readily sealed onto patch pipettes and with pipette solutions containing nicotinic agonists, single channel currents could be recorded. With cell-attached patches, these currents were inward at the cell resting potential and reversed with depolarization. Channels with two different conductances could be seen in the majority of patches.