UK-414,495, a selective inhibitor of neutral endopeptidase, potentiates pelvic nerve-stimulated increases in female genital blood flow in the anaesthetized rabbit.

BACKGROUND AND PURPOSE: Female sexual arousal consists of a number of physiological responses resulting from increased genital blood. Vasoactive intestinal peptide (VIP), neuropeptide Y and to a lesser extent nitric oxide are neurotransmitters found in the vasculature of the genitalia. Neutral endopeptidase (NEP) modulates the activity of neuropeptides including VIP. The aim of this study was to investigate the control of genital blood flow by VIP and endogenous neuropeptides using a selective NEP inhibitor [UK-414,495, ((R)-2-({1-[(5-ethyl-1,3,4-thiadiazol-2-yl) carbamoyl]cyclopentyl}methyl) valeric acid)]. EXPERIMENTAL APPROACH: Vaginal and clitoral blood flow (VBF and CBF) were monitored using laser Doppler in terminally anaesthetized New Zealand rabbits. Increases in VBF and CBF were induced by either electrical stimulation of the pelvic nerve or by i.v. infusion of VIP. KEY RESULTS: Stimulation of the pelvic nerve increased VBF and CBF, compared with basal flow. Increases were mimicked by infusion of exogenous VIP. UK-414,495 dose-dependently potentiated pelvic nerve-stimulated increases in VBF (EC(50)= 37 +/- 9 nM; 3.6 x IC(50) rabbit NEP). Nerve-stimulated increases in VBF and CBF were both enhanced after UK-414,495. UK-414,495 increased the amplitude and duration of VIP-induced increases in VBF. UK-414,495 had no effect on basal VBF or cardiovascular parameters. CONCLUSIONS AND IMPLICATIONS: Inhibition of NEP potentiates pelvic nerve-stimulated increases in genital blood flow. This suggests that the endogenous neurotransmitter mediating genital blood flow is a substrate for NEP (most likely VIP). NEP inhibitors may restore sexual arousal in women adversely affected by female sexual arousal disorder.

Dopamine-oxytocin interactions in penile erection.

Dopamine and oxytocin have established roles in the central regulation of penile erection in rats; however, the neural circuitries involved in a specific erectile context and the interaction between dopamine and oxytocin mechanisms remain to be elucidated. The medial preoptic area (MPOA), supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus may serve as candidate sites because they contain oxytocin cells, receive dopaminergic inputs and have been implicated in mediating masculine sexual behavior. Double immunofluorescence revealed that substantial numbers of oxytocin cells in the MPOA, SON and PVN possess dopamine D(2), D(3) and D(4) receptors. In anaesthetized rats, using intracavernous pressure as a physiological indicator of erection, blockade of lumbosacral oxytocin receptors (UK, 427843) reduced erectile responses to a nonselective dopamine agonist (apomorphine), suggesting that dopamine recruits a paraventriculospinal oxytocin pathway. In conscious males in the absence of a female, penile erection elicited by a D(2)/D(3) (Quinelorane) but not D(4) (PD168077) agonist was associated with activation of medial parvocellular PVN oxytocin cells. In another experiment where males were given full access to a receptive female, a D(4) (L-745870) but not D(2) or D(3) antagonist (L-741626; nafadotride) inhibited penile erection (intromission), and this was correlated with SON magnocellular oxytocin neuron activation. Together, the data suggest dopamine's effects on hypothalamic oxytocin cells during penile erection are context-specific. Dopamine may act via different parvocellular and magnocellular oxytocin subpopulations to elicit erectile responses, depending upon whether intromission is performed. This study demonstrates the potential existence of interaction between central dopamine and oxytocin pathways during penile erection, with the SON and PVN serving as integrative sites.

Pharmacological profiling of neuropeptides on rabbit vaginal wall and vaginal artery smooth muscle in vitro.

BACKGROUND AND PURPOSE: Hypothalamic neuropeptides centrally modulate sexual arousal. However, the role of neuropeptides in peripheral arousal has been ignored. Vascular and non-vascular smooth muscle relaxation in the vagina is important for female sexual arousal. To date, in vitro studies have focused on vaginal strips with no studies on vaginal arteries. The aim of this study was to compare the effects of sexual hypothalamic neuropeptides on rabbit vaginal wall strips and arteries. EXPERIMENTAL APPROACH: Tissue bath and wire myography techniques were used to measure isometric tension from strips and arteries, respectively. KEY RESULTS: Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) relaxed both preparations, effects that were only antagonized by the VIP/PACAP antagonist VIP6-28 (10 nM) and the PAC(1) antagonist PACAP 6-38 (1 microM). The melanocortin agonist alpha-melanocortin-stimulating hormone (1 microM), but not bremelanotide (1 microM), also relaxed both preparations. Oxytocin and vasopressin contracted vaginal preparations, which could be antagonized by the V(1A) antagonist SR 49059. Neuropeptide Y (NPY) and the NPY Y(1) agonist Leu(31), Pro(34) NPY only contracted arteries, which was antagonized by the NPY Y(1) receptor antagonist BIBP 3226. Melanin-concentrating hormone (MCH; 1 microM) contracted arteries. CONCLUSION AND IMPLICATIONS: Hypothalamic neuropeptides can exert contractile and relaxant effects on vaginal strips and arteries. NPY Y(1), V(1A), MCH(1) antagonists as well as VIP/PAC(1) agonists may have therapeutic potential in both central and peripheral female sexual arousal. Differences in effect of neuropeptides between preparations raise the question of which preparation is important for female sexual arousal.

Phosphodiesterase 11 (PDE11) regulation of spermatozoa physiology.

Fertilization is well correlated with sperm concentration, rate of forward motility, and percentage of live, uncapacitated ejaculated spermatozoa, which is regulated in part by cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to their corresponding monophosphates, thereby counterbalancing the activities of cAMP and cGMP, and PDE11 is highly expressed in the testis, prostate, and developing spermatozoa. However, a physiological role of PDE11 is not known. We generated PDE11 knockout (PDE11-/-) mice to investigate the role of PDE11 in spermatozoa physiology. Ejaculated sperm from PDE11-/- mice displayed reduced sperm concentration, rate of forward progression, and percentage of live spermatozoa. Pre-ejaculated sperm from PDE11-/- mice displayed increased premature/spontaneous capacitance. These data are consistent with human data and suggest a role for PDE11 in spermatogenesis and fertilization potential. This is the first phenotype described for the PDE11-/- mouse and the first report of a physiological role for PDE11.

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.

Hospital-based nursing case management: role clarification.

Hospital-based nursing case management as a model of healthcare delivery has substantially grown over the last 10 years. Nursing case management as a viable professional role has developed along with this trend. In an era of decreasing reimbursements and increasing accreditation requirements, hospital administrators view nurse case managers as one answer to balancing cost and quality. However, the actual role and practice of nurse case managers within the hospital setting is inconsistent and often depends on the needs and expectations of the organization, as well as the level of experience and educational preparation of the nurse case manager.

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.

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.

N-methyl-D-aspartate (NMDA) stimulates release of alpha-MSH from the rat hypothalamus through release of nitric oxide.

Superfusion of rat hypothalamic slices with 10(-4) M N-methyl-D-aspartic acid (NMDA) resulted in increased release of alpha-melanocyte-stimulating hormone (alpha-MSH). Peptide release was blocked by 10(-6) M NG-nitro-L-arginine methyl ester (L-NAME) a specific competitive inhibitor of nitric oxide synthase but not by the inactive enantiomer D-NAME at 10(-6) M. The inhibition by L-NAME was reversed by the addition of 10(-5) mM L-arginine, an excess of enzyme substrate. Release of nitric oxide products into tissue superfusates was stimulated by a 50 mM concentration of potassium ions and by 10(-4) M NMDA. Potassium-stimulated release was blocked by L-NAME. Basal, potassium-stimulated and NMDA-stimulated release of nitric oxide products were significantly inhibited by the NMDA-receptor antagonist D(-)-2-amino-5-phosphopentanoic acid (AP5) at 10(-4) M and by the NMDA-channel blocker ketamine at 10(-4) M. We conclude that nitric oxide mediates the stimulatory action of glutamic acid on the release of alpha-MSH from the rat hypothalamus.

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.

Endogenous glutamate stimulates release of alpha-melanocyte-stimulating hormone from the rat hypothalamus.

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) and glutamic acid was quantified from superfused slices of rat hypothalamus. Application of L-glutamic acid 10(-4) M failed to evoke release of alpha-MSH but, in the presence of 10(-4) M dihydrokainic acid (DHK) an inhibitor of glutamate uptake systems, caused significant stimulation of release. DHK caused gradual and sustained increases in both alpha-MSH and glutamate release. That in alpha-MSH was blocked by 10(-4) M DL-2-amino-5-phosphopentanoic acid, a competitive N-methyl-D-aspartic acid (NMDA)-type glutamate receptor antagonist. We conclude that hypothalamic glutamate is subject to rapid uptake through mechanisms blocked by DHK and that alpha-MSH release is stimulated by endogenous and exogenous glutamate through NMDA-type receptors.

The role of endoscopy after vertical banded gastroplasty.

Since 1984, a total of 99 patients underwent vertical banded gastroplasty (VBG) through protocol (pouch 8 ml in size, band 4.3 cm in circumference) to treat morbid obesity. Follow-up was obtained in 95 patients. Thirty upper gastrointestinal endoscopies were performed post-operatively in 17 patients. Indications were nausea/vomiting in 11, epigastric pain in 4, acute obstructive symptoms in 4, and miscellaneous in three. Findings included food impaction in 10, distal esophagitis in 8, gastritis in 4, and a normal examination in 2. Only 4 of 10 food impactions were associated with an excessively narrowed gastroplasty outlet. Eight patients had an excessively narrowed gastric stoma: two became asymptomatic with dietary modification only and six underwent dilation therapy (dilator range from 8 to 18 mm in diameter) with immediate resolution of symptoms in four of six. One of the two patients unresponsive to dilation was lost to follow-up, and the other required surgical revision after multiple dilation sessions.

Endogenous gamma-aminobutyric acid tonically inhibits release of alpha-melanocyte-stimulating hormone from rat hypothalamic slices.

Release of immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) from superfused slices of rat hypothalamus was stimulated by the gamma-aminobutyric acid (GABA) receptor antagonist, bicuculline, and inhibited by the benzodiasepine, chlordiazepoxide, an allosteric GABA receptor modulator, demonstrating the presence of tonic inhibition of alpha-MSH release by endogenous GABA in hypothalamic tissue. Chlordiazepoxide increased the effect of exogenous GABA which by inhibiting basal release of alpha-MSH demonstrated that the tonic inhibition was not maximal in the resting state.

Characteristics of NMDA receptors that stimulate release of hypothalamic alpha-MSH.

N-methyl-D-aspartic acid (NMDA) 10(-4) M stimulated release of immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) from superfused slices of rat hypothalamus through receptors which shared common features with other central NMDA-type glutamate receptors. The receptors possessed inhibitory sites for both Mg2+ and ketamine; basal and NMDA-stimulated alpha-MSH release was reduced by high (5 mM) Mg2+ ion concentrations and by 10(-4) M ketamine, whilst use of Mg(2+)-free media led to a prolongation of the NMDA-stimulated response. The receptors were also shown to possess an allosteric glycine site. The glycine site agonist D-serine 10(-4) M potentiated basal and NMDA-stimulated alpha-MSH release whilst the antagonist, 7-chlorokynurenic acid 10(-4) M, reduced NMDA-stimulated release, an effect which was partially reversed by 10(-4) M D-serine.