The expression of a cloned Drosophila octopamine/tyramine receptor in Xenopus oocytes.

The expression of a cloned Drosophila octopamine/tyramine receptor (OctyR99AB) is described in Xenopus oocytes. Agonist stimulation of OctyR99AB receptors increased intracellular Ca2+ levels monitored as changes in the endogenous inward Ca2+-dependent chloride current. The receptor is preferentially sensitive to biogenic amines with a single hydroxyl on the aromatic ring. The G-protein, Galphai, appears to be involved in the coupling of the receptor to the production of intracellular calcium signals, since the effect is pertussis-toxin sensitive and is blocked or substantially reduced in antisense knockout experiments using oligonucleotides directed against Galphai but not by those directed against Galphao, Galphaq and Galpha11. The increase in intracellular calcium levels induced by activation of the OctyR99AB receptor can potentiate the ability of activation of a co-expressed beta2-adrenergic receptor to increase oocyte cyclic AMP levels. A comparison of the pharmacological coupling of OctyR99AB to different second messenger systems when expressed in Xenopus oocytes with previous studies on the expression of the receptor in a Chinese hamster ovary cell line suggests that the property of agonist-specific coupling of the receptor to different second messenger systems may be cell-specific, depending upon the G-protein environment of any particular cell type.

A comparison of the release of a vasoactive-intestinal-peptide-like peptide and acetylcholine in the giant axon-Schwann cell preparation of the tropical squid Sepioteuthis sepioidea.

A vasoactive intestinal peptide (VIP)-like peptide is released by axonal stimulation in the giant axon-Schwann cell preparation from the tropical squid Sepioteuthis sepioidea. It is also released by direct application of l-glutamate, the giant axon-Schwann cell signalling molecule in this preparation. The release of the peptide parallels the release of acetylcholine from the Schwann cells themselves in this preparation in a number of different ways. The release of both acetylcholine and the VIP-like peptide have the same threshold (between 2x10(-10) and 5x10(-10 )mol l-1) for l-glutamate application and the same recovery time after inhibition of release by exposure of the preparation to a prolonged pulse of l-glutamate. A prolonged l-glutamate pulse of 10(-8 )mol l-1 releases both substances for as long as the pulse is applied to the preparation, whereas a prolonged pulse of 10(-9 )mol l-1 l-glutamate releases acetylcholine in the same way but releases the VIP-like peptide only transiently. The VIP-like peptide is likely to be co-released with acetylcholine from the Schwann cells.

Vasoactive intestinal peptide (VIP) induces a K+ current in the Xenopus oocyte membrane.

Evidence, based on voltage-clamp and current-clamp recording, is presented for endogenous receptors for vasoactive intestinal polypeptide (VIP) on the cell membrane of the Xenopus oocyte. At normal resting potential a hyperpolarisation is produced by VIP, associated with an increase in K+ conductance. The response is dose-dependent, with a threshold near 10(-9)M VIP. The effect is potentiated by forskolin. Of several other types of neuropeptide tested, only avian pancreatic polypeptide gives a response: this is similar to that of VIP, but this peptide can also potentiate the response to VIP.

Comparison of the operating microscope and loupes for free microvascular tissue transfer.

A retrospective study of 200 consecutive free microvascular tissue transfers over a 3-year period was done to compare the performance of free-tissue transfers with loupes and with the operating microscope. One-hundred and nineteen flaps (59.5 percent) were performed under 3.5 x loupe magnification and 81 under the operating microscope (40.5 percent). The magnification selection process was based on cumulative past experience, with all early anastomoses performed with the microscope and the vast majority of the more recent operations performed with loupe magnification. Loupes were used preferentially for head and neck reconstruction and breast reconstruction. The microscope was required for performing vascular anastomoses on children and on vessels less than or equal to 1.5 mm in diameter. Results were compared with respect to etiology of defect, type of flap, age of patient, free-flap success, complications, and overall success of the reconstruction. There was no difference in outcome between the two groups, with free-flap success rates of 99 percent for both the loupe and the microscope groups. We believe that our success with loupe-only free-tissue transfers is attributable to our prior considerable experience with the microscope. We would caution that comfort and experience with microanastomoses under the operating microscope should be obtained prior to beginning a loupe-only experience.

Peptidergic modulation of the membrane potential of the Schwann cell of the squid giant nerve fibre.

The effects of a range of neuropeptides were investigated on the membrane potential of the Schwann cells of the giant nerve fibre of the tropical squid. Vasoactive intestinal peptide (VIP) produced a dose-dependent, long-lasting hyperpolarization of the Schwann-cell membrane potential. Among peptides structurally related to VIP, similar effects were produced by peptide histidine isoleucine (PHI) but not by secretin and glucagon. Substance P and somatostatin also hyperpolarized the Schwann-cell membrane potential but via receptor systems distinct from those activated by VIP. Methionine enkephalin ([Met]-enkephalin) blocked the actions of all the above peptides as well as the effects of DL-octopamine and carbachol. The actions of [Met]-enkephalin upon the VIP responses were antagonized by naloxone. VIP produces its effects on the Schwann-cell membrane potential via a receptor system that is independent from those described previously which mediate the effects of carbachol and DL-octopamine. However, VIP can potentiate the effects of the latter systems. The actions of VIP on the Schwann cell are unlikely to be mediated via changes in adenosine 3',5'-cyclic monophosphate (cyclic AMP) levels and are insensitive to changes in the level of extracellular calcium in the superfusate. The actions of VIP are, however, potentiated in the presence of low concentrations of lithium ions suggesting that the VIP receptor may mediate its effects by inducing the hydrolysis of polyphosphatidylinositols in the Schwann-cell membrane. Evidence is presented for the existence of an endogenous VIP-like component in the normal hyperpolarizing action of giant-axon activity on the membrane potential of the Schwann cell.

Octopaminergic modulation of the membrane potential of the Schwann cell of the squid giant nerve fibre.

The actions of octopamine on the Schwann cells of the giant nerve fibre of the tropical squid are described. The pharmacology of the receptors mediating the actions of octopamine has been investigated in terms of stereospecificity, amine specificity and interactions with a range of agonists and antagonists. The receptors are maximally activated by D(-)-octopamine and share many of the characteristics of OCTOPAMINE2 class receptors in other preparations. The octopamine receptors appear to mediate their actions by increasing the intracellular levels of cyclic AMP in the Schwann cells. Low concentrations of octopamine potentiate the actions of the nicotinic cholinergic activation system of the Schwann cells. The results are discussed in terms of the possible physiological role of octopamine in the modulation of Schwann cell activity during stressful conditions when the giant axon system is likely to be used at a high frequency to facilitate the escape response of the squid.

The role of cyclic nucleotides in modulation of the membrane potential of the Schwann cell of squid giant nerve fibre.

The role of cyclic nucleotides in mediating the effects of nicotine cholinergic receptors has been investigated in Schwann cells of the giant nerve fibre of the squid. Elevation of cyclic AMP levels in this preparation by means of the phosphodiesterase inhibitor, theophylline, by the diterpene adenylate cyclase activator, forskolin, and by cyclic nucleotide analogues mimics the action of activating the nicotinic cholinergic receptors in producing a long-lasting hyperpolarization of the membrane potential of the Schwann cell. Theophylline and forskolin also potentiate the effects of carbachol and of neural stimulation on the Schwann cell. The results suggest that the nicotinic receptor of the squid Schwann cell is likely to mediate its effects via a mechanism that activates adenylate cyclase. The results are discussed in terms of the role of cyclic AMP in the complex multistep interaction between the giant axon of the squid and its surrounding Schwann-cell layer.

Modulation of the membrane potential of the Schwann cell of the squid giant nerve fiber.

1. The involvement of second messengers and of other chemical mediators, in the modulation of the membrane potential of the Schwann cell of the giant nerve fiber of the Tropical squid Sepioteuthis sepioidea is described. 2. The involvement of the cyclic nucleotide adenosine 3', 5' monophosphate (cAMP) in mediating the actions of the nicotinic Ach receptors of the Schwann cells is suggested. 3. The presence of octopaminergic receptors in the Schwann cells, mediating their actions through the activation of adenylate cyclase, is also described. 3. Receptors for vasoactive intestinal peptide (VIP) are also present on the Schwann cells, and their actions are mediated via a second messenger system that does not involve the activation of adenylate cyclase. 5. The three independent receptor systems referred above are able to interact in a complex way, which involves both their direct actions on the Schwann cell membrane potential and modulatory effects between the systems.

Sweat gland carcinoma of the foot: a review and patient report.

Sweat gland neoplasms, whether benign or malignant, are rare. A case of sweat gland carcinoma occurring in the foot is represented. The literature, including clinical presentation, dermatopathology, and treatment is reviewed.

Nucleotide Regulation of a calcium-activated cation channel in the rat insulinoma cell line, CRI-G1.

The nucleotide regulation of a calcium-activated nonselective cation (Ca-NS+) channel has been investigated in the rat insulinoma cell line CRI-G1. The activity of the channel is reduced by both AMP and ADP (1-100 microM) in a concentration-dependent manner, with AMP being more potent than ADP. At lower concentrations (0.1-5 microM), both ADP and AMP activate the channel in some patches. Examination of the nucleotide specificity of channel inhibition indicates a high selectivity for AMP over the other nucleotides tested with a rank order of potency of AMP > UMP > CMP > or = GMP. Cyclic nucleotides also modulate channel activity in a complex, concentration-dependent way. Cyclic AMP exhibits a dual effect, predominantly increasing channel activity at low concentrations (0.1-10 microM) and reducing it at higher concentrations (100 microM and 1 mM). Specificity studies indicate that the cyclic nucleotide site mediating inhibition of channel activity exhibits a strong preference for cyclic AMP over cyclic GMP, with cyclic UMP being almost equipotent with cyclic AMP. Cyclic IMP and cyclic CMP are not active at this site. The cyclic nucleotide site mediating activation of the channel shows much less nucleotide specificity than the inhibitory site, with cyclic AMP, cyclic GMP and cyclic IMP being almost equally active.

The effects of pyridine nucleotides on the activity of a calcium-activated nonselective cation channel in the rat insulinoma cell line, CRI-G1.

The activity of a calcium-activated nonselective (Ca-NS+) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, beta-NAD+, beta-NADH, beta-NADP+ and beta-NADPH were very similar when tested at 0.1 mM, and at 1 mM the phosphorylated forms, beta-NADP+ and beta-NADPH, appeared to be slightly more potent than beta-NAD+ and beta-NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch. The application of beta-NAD+, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the beta-NAD(+)-NS+ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP.

Regulation of calcium-activated nonselective cation channel activity by cyclic nucleotides in the rat insulinoma cell line, CRI-G1.

The regulation of a calcium-activated nonselective cation (Ca-NS+) channel by analogues of cyclic AMP has been investigated in the rat insulinoma cell line, CRI-G1. The activity of the channel is modulated by cyclic AMP in a complex way. In the majority of patches (83%) tested concentrations of cyclic AMP of 10 microM and above cause an inhibition of channel activity which is immediately reversible on washing. In contrast, lower concentrations of cyclic AMP, between 0.1 and 1.0 microM, produce a transient activation of channel activity in most patches (63%) tested. One group of analogues, including N6-monobutyryl cyclic AMP and N6, 2'-O-dibutyryl cyclic AMP reduced the activity of the Ca-NS+ channel at all concentrations tested and 2'-O-Monobutyryl cyclic AMP produced inhibition in all patches tested except one, at all concentrations. A second group produced dual concentration-dependent effects on Ca-NS+, low concentrations stimulating and high concentrations inhibiting channel activity. 6-Chloropurine cyclic AMP and 8-bromo cyclic AMP produced effects similar to those of cyclic AMP itself. In contrast, 8-[4-chlorophenylthio] cyclic AMP also showed a dual action, but with a high level of activation at all concentrations tested up to 1 mM. Ca-NS+ channel activity was also predominantly activated by low concentrations of Sp-cAMPS. The activating effects of both Sp-cAMPS and cyclic AMP are antagonized by Rp-cAMPS, which by itself only produced a weak inhibition of Ca-NS+ channel activity even at concentrations of 10 microM and above. The results are discussed in terms of a model in which cyclic AMP, and other cyclic nucleotides, modulate the activity of the Ca-NS+ channel by binding to two separate sites.

[Neuron-neuroglia functional relation in the squid giant nerve fiber]. / Relaciones functionales neurona-neuroglia en la fibra nerviosa gigante del calamar.

The present work reviews the results of a series of physiological studies carried out in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea, aimed at exploring the possible involvement of different second mediators and receptor systems, in the modulation of the long-lasting hyperpolarizing response of the Schwann cell membrane potential to axonal excitation. These studies provide experimental evidence on the existence of distinct receptors for ACh, octopamine, and a VIP-like endogenous peptide in the Schwann cell plasma membrane. They also indicate that the cyclic nucleotide adenosine 3',5' monophosphate may be playing a role as second messenger for the actions of the Schwann cell nicotinic cholinergic and the octopaminergic receptors, whereas the VIP receptor may be acting through the hydrolysis of polyphosphatidylinositols. These experimental findings are discussed as evidence for the existence of a complex multistep interaction between the giant axon and its satellite glial cells, the Schwann cell.

Agonist-specific coupling of a cloned Drosophila melanogaster D1-like dopamine receptor to multiple second messenger pathways by synthetic agonists.

The mechanism of coupling of a cloned Drosophila D1-like dopamine receptor, DopR99B, to multiple second messenger systems when expressed in Xenopus oocytes is described. The receptor is coupled directly to the generation of a rapid, transient intracellular Ca2+ signal, monitored as changes in inward current mediated by the oocyte endogenous Ca2+-activated chloride channel, by a pertussis toxin-insensitive G-protein-coupled pathway. The more prolonged receptor-mediated changes in adenylyl cyclase activity are generated by an independent G-protein-coupled pathway that is pertussis toxin-sensitive but calcium-independent, and Gbetagamma-subunits appear to be involved in the transduction of this response. This is the first evidence for the direct coupling of a cloned D1-like dopamine receptor both to the activation of adenylyl cyclase and to the initiation of an intracellular Ca2+ signal. The pharmacological profile of both second messenger effects is identical for a range of naturally occurring catecholamine ligands (dopamine > norepinephrine > epinephrine) and for the blockade of dopamine responses by a range of synthetic antagonists. However, the pharmacological profiles of the two second messenger responses differ for a range of synthetic agonists. Thus, the receptor exhibits agonist-specific coupling to second messenger systems for synthetic agonists. This feature could provide a useful tool in the genetic analysis of the roles of the multiple second messenger pathways activated by this receptor, given the likely involvement of dopamine in the processes of learning and memory in the insect nervous system.

N-methyl-D-aspartate (NMDA) and non-NMDA (metabotropic) type glutamate receptors modulate the membrane potential of the Schwann cell of the squid giant nerve fibre.

L-Glutamate application can produce three different responses in the membrane potential of the Schwann cell of the tropical squid, Sepioteuthis sepioidea, which appear to be mediated by three pharmacologically distinct classes of receptor. A class of non-NMDA-type receptors, with some similarities to metabotropic glutamate receptors, mediates the development of a rapid and long-lasting hyperpolarization. Two pharmacologically distinct classes of NMDA-type receptor are present. One mediates the development of a slow depolarization accompanied by a long-lasting change in responsiveness of the Schwann cell. The second produces rapid depolarizing responses during the period of this changed responsiveness. All three types of receptor can be activated by dipeptides containing excitatory amino acids.

The effect of a glutamate uptake inhibitor on axon-Schwann cell signalling in the squid giant nerve fibre.

The glutamate uptake blocker p-chloromercuriphenylsulphonic acid (PCMS) (100 mumol l-1) does not block any of the membrane potential changes induced by the application of L-glutamate to the adaxonal Schwann cells of the giant axon of the tropical squid Sepioteuthis sepioidea. This indicates that these potential changes are not due to the activation of an electrogenic glutamate uptake system and supports the idea that they are due to the activation of specific glutamate receptors. The presence of PCMS (100 mumol l-1) reduces the activity of the glutamate uptake system sufficiently for the extracellular level of axonally released glutamate to exceed the threshold for the activation of the NMDA-type glutamate receptors in this preparation.