Plexus Of Guinea-pig Small Intestine Research Articles

P.1.c.005 Polymorphism of the serotonin transporter promoter may predict the response to selective serotonin reuptake inhibitors in bulimia nervosa

Conventional intracellular microelectrodes and marker injection techniques were used to study the actions of histamine on inhibitory synaptic transmission in the submucous plexus of guinea-pig small intestine. Bath application of histamine (1–300 μM) reversibly suppressed both noradrenergic and non-adrenergic slow inhibitory postsynaptic potentials in a concentration-dependent manner. These effects of histamine were mimicked by the selective histamine H3 receptor agonist R(−)-α-methylhistamine but not the selective histamine H1 receptor agonist, 6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl) heptanecarboxamide (HTMT dimaleate), or the selective histamine H2 receptor agonist, dimaprit. The histamine H3 receptor antagonist, thioperamide, blocked the effects of histamine. Histamine H1 and H2 receptor antagonists did not change the action of histamine. Hyperpolarizing responses to focal application of norepinephrine or somatostatin by pressure ejection from micropipettes were unaffected by histamine and R(−)-α-methylhistamine. The results suggest that histamine acts at presynaptic histamine H3 receptors on the terminals of sympathetic postganglionic fibers and intrinsic somatostatinergic nerves in the small intestine to suppress the release of the inhibitory neurotransmitters, norepinephrine and somatostatin.

Slow excitatory synaptic transmission mediated by P2Y1 receptors in the guinea-pig enteric nervous system.

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y1 receptors on morphologically identified neurones in the submucosal plexus of guinea-pig small intestine. MRS2179, a selective P2Y1 purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S-type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y1 receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5-trisphosphate and mobilization of Ca2+ from intracellular stores.

Actions of bradykinin on electrical and synaptic behavior of neurones in the myenteric plexus of guinea-pig small intestine.

1. Electrophysiologic methods were used to study actions of bradykinin (BK) in neurones of the myenteric plexus of guinea-pig small intestine in vitro. Exposure to BK depolarized the membrane potential and elevated excitability in AH- and S-type neurones. Neuronal input resistance associated with the depolarizing responses was either decreased or unchanged in S-type and increased in AH-type neurones. 2. The selective B(2) BK receptor antagonist HOE-140, but not the selective B(1) receptor antagonist des-arg(10)-HOE-140, suppressed the BK-evoked responses. RT-PCR confirmed the expression of B(2) receptor mRNA, but not B(1) receptor mRNA. 3. Binding of fluorescently- labeled HOE-140 (HOE741) was localized to ganglion cells in whole-mount preparations. BK B(2) receptors were coexpressed with immunoreactivity for calbindin or nitric oxide synthase. 4. Exposure to BK suppressed the amplitude of both fast and slow excitatory postsynaptic potentials. Depolarizing responses evoked by application of serotonin or substance P and nicotinic responses to acetylcholine were not reduced by BK. This suggested that BK action on neurotransmission was presynaptic suppression of neurotransmitter release. Presence of HOE-140 in the bathing solution suppressed or abolished the presynaptic inhibitory action of BK. 5. The cyclooxygenase inhibitor, piroxicam, suppressed both the direct excitatory action of BK and its presynaptic inhibitory action. Application of prostaglandin E(2), D(2), F(2alpha) or I(2) mimicked the BK-evoked responses. 6. The results suggest that BK acts at B(2) BK receptors on myenteric neurones to stimulate the formation of prostaglandins. Once formed and released, the prostaglandins act to elevate the excitability of ganglion cells in the myenteric plexus and to suppress the synaptic release of neurotransmitters.

Immunoreactivity of Hu proteins facilitates identification of myenteric neurones in guinea-pig small intestine.

Hu proteins, together with neurone-specific enolase (NSE), protein gene product 9.5 (PGP-9.5), microtubule-associated protein-2 (MAP-2) and tubulin beta III isoform, were evaluated immunohistochemically as neuronal markers in whole-mount preparations and cultures obtained from the myenteric plexus of guinea-pig small intestine. Anti-Hu immunostaining marked the ganglion cell somas and nuclei without staining of the neuronal processes in the whole-mounts and cultures. The ganglion cell bodies were not obscured by staining of multiple neuronal fibres and this facilitated accurate counting of the neurones. MAP2 immunostaining also provided clear images of individual neurones in both whole mounts and cultures. Immunoreactivity for NSE, PGP-9.5 and tubulin beta III isoform provided sharp images of the ganglion cells in culture, but not in whole-mount preparations. Strong staining of the neuronal processes in the whole-mount preparations obscured the profiles of the ganglion cell bodies to such an extent that accurate counting of the total neuronal population was compromised. Anti-Hu immunostaining was judged to be an acceptable method for obtaining reliable estimates of total numbers of myenteric neurones in relation to other specific histochemical properties such as histamine binding.

Sodium conductance in cultured myenteric AH-type neurons from guinea-pig small intestine

Whole-cell patch clamp methods were used to investigate sodium conductance in after-hyperpolarization-type (AH) enteric neurons in culture after dissociation from the myenteric plexus of guinea-pig small intestine. Inward current carried by Na + ( I Na) was identified and its current–voltage characteristics were compared with those for inward Ca 2+ current ( I Ca). The I Na current was a rapidly inactivating current relative to I Ca. Application of tetrodotoxin (TTX) blocked I Na with an EC 50 of 10.7 nM. Activation curves for I Na showed a rapid decrease in time to peak for test potentials from holding potentials of −80 mV to between −40 and −10 mV. Voltage-dependence of steady-state inactivation curves for I Na was fit to the Boltzmann equation with potential for half-inactivation ( V 1/2)=−55.6 mV and slope factor ( k)=6.4 mV. Steady-state inactivation for I Ca fit the Boltzmann equation with a V 1/2=−38.9 mV and k=14.4 mV. Kinetics for inactivation of I Na were voltage dependent at potentials between −70 and −30 mV and accelerated and became less voltage-dependent at more positive potentials. The time constant ( τ) for inactivation at −70 mV was τ=161±23 ms and decreased to τ=2.3±0.2 ms at −30 mV. Rapid acceleration of inactivation occurred between −50 and −40 mV. This was also the range where activation began. Recovery from inactivation with the membrane potential clamped at −100 or −80 mV was rapid and fit by a single exponential with τ=7.3±1.1 ms for −100 mV and 21.5±5.1 ms for −80 mV. The results suggest that AH-type enteric neurons have only one type of Na + channel that behaves like the “classical” voltage-gated tetrodotoxin-sensitive fast channel. The findings support the hypothesis that I Na current is an important factor in determination of excitability and firing behavior in AH neurons. I Na and I Ca together determine the properties of the rising phase of the spike and thereby contribute to global determinants of excitability as the neurons are exposed to multiple depolarizing and hyperpolarizing stimuli from synaptic inputs and mediators released from enteroparacrine cells.

A novel metabotropic P2Y, receptor in the submucous plexus of guinea-pig small intestine

A novel metabotropic P2Y, receptor in the submucous plexus of guinea-pig small intestine

A novel metabotropic P2Y, receptor in the submucous plexus of guinea-pig small intestine

A novel metabotropic P2Y, receptor in the submucous plexus of guinea-pig small intestine

Glutamate receptors in the enteric nervous system: ionotropic or metabotropic?

Intracellular recording methods were used to investigate actions of glutamate on morphologically identified neurones in the myenteric and submucous plexuses of guinea-pig small intestine. Glutamate evoked a tetrodotoxin-resistant, slowly activating depolarizing response in most of the submucous neurones (86 of 125, 69%) and a smaller number of myenteric neurones (6 of 60, 10%). The depolarizing responses were restricted to S-type neurones with uniaxonal morphology. The group I metabotropic glutamate receptor (mGluRs) agonists quisqualate, 1S, 3R-ACPD and DHPG mimicked the depolarizing action of glutamate. A group I mGluRs antagonist, S-4-carboxyphenylglycine (S-4CPG), suppressed the glutamate responses with an IC50 of 357 microM at 30 microM glutamate. Group II or III mGluRs agonists did not produce depolarizing responses and group II or III mGluRs antagonists did not alter glutamate-evoked depolarization. The ionotropic glutamate receptor (iGluRs) agonists NMDA, AMPA, or kainate did not evoke depolarizing responses and glutamate-evoked depolarization was unaffected by the iGluRs antagonists D-APV, MK-801, or DNQX. No rapidly activating fast depolarizing responses reminiscent of fast excitatory postsynaptic potentials (EPSPs) were ever observed during application of glutamate or AMPA and stimulus-evoked fast EPSPs were unaffected by DNQX. The results suggest that the excitatory action of glutamate on enteric neurones is mediated by group I metabotropic glutamate receptors and that ionotropic glutamate receptors are not involved. The results also suggest that glutamate-mediated fast EPSPs may not be present in myenteric and submucous neurones in guinea-pig small bowel.

Histamine H 3 receptor-mediated suppression of inhibitory synaptic transmission in the submucous plexus of guinea-pig small intestine

Conventional intracellular microelectrodes and marker injection techniques were used to study the actions of histamine on inhibitory synaptic transmission in the submucous plexus of guinea-pig small intestine. Bath application of histamine (1–300 μM) reversibly suppressed both noradrenergic and non-adrenergic slow inhibitory postsynaptic potentials in a concentration-dependent manner. These effects of histamine were mimicked by the selective histamine H 3 receptor agonist R(−)-α-methylhistamine but not the selective histamine H 1 receptor agonist, 6-[2-(4-imidazolyl)ethylamino]- N-(4-trifluoromethylphenyl) heptanecarboxamide (HTMT dimaleate), or the selective histamine H 2 receptor agonist, dimaprit. The histamine H 3 receptor antagonist, thioperamide, blocked the effects of histamine. Histamine H 1 and H 2 receptor antagonists did not change the action of histamine. Hyperpolarizing responses to focal application of norepinephrine or somatostatin by pressure ejection from micropipettes were unaffected by histamine and R(−)-α-methylhistamine. The results suggest that histamine acts at presynaptic histamine H 3 receptors on the terminals of sympathetic postganglionic fibers and intrinsic somatostatinergic nerves in the small intestine to suppress the release of the inhibitory neurotransmitters, norepinephrine and somatostatin.

Histamine H 3 receptor-mediated suppression of inhibitory synaptic transmission in the submucous plexus of guinea-pig small intestine

Histamine H 3 receptor-mediated suppression of inhibitory synaptic transmission in the submucous plexus of guinea-pig small intestine

Functional group I metabotropic glutamate receptors in submucous plexus of guinea‐pig ileum

Intracellular recording methods and immunostaining revealed the existence of functional group I metabotropic glutamate receptors (mGluRs) in submucous plexus neurons of guinea-pig ileum. Selective group I, but not groups II or III metabotropic glutamate receptor agonists induced concentration-dependent, slowly-activating depolarizing responses. Group I metabotropic glutamate receptor antagonism observed with (S)-4-carboxyphenylglycine (S-4-CPG) (100 - 600 microM) was competitive as determined by Schild analysis (pA(2)=3.81+/-0.02). Neither the group II and III metabotropic nor ionotropic glutamate receptor antagonists altered responses evoked by group I receptor agonists. Immunoreactivities for metabotropic glutamate 1alpha and 5 receptors were found to locate exclusively in neurons in the submucous plexus of guinea-pig ileum with the highest density around the cell bodies. The results suggest that group I metabotropic glutamate receptors are functionally expressed in the submucous plexus of guinea-pig small intestine.

Glutamate modulates neurotransmission in the submucosal plexus of guinea-pig small intestine.

Effects of glutamate on synaptic transmission in the submucosal plexus of guinea-pig small intestine were studied with intracellular electrophysiological recording methods. Glutamate suppressed stimulus-evoked slow excitatory postsynaptic potentials (EPSPs) and increased the amplitude of slow inhibitory postsynaptic potentials (IPSPs) in submucosal neurons. The actions of glutamate were mimicked by the group I metabotropic glutamate receptor (mGluRs) agonist DHPG, but not by the group II agonist S-4C3HPG, the group III agonist L-AP4, or selective agonists for ionotropic glutamate receptors (iGluRs). Glutamate actions were suppressed by the selective group I mGluRs antagonist S-4CPG, but not by group II and III mGluRs antagonist CPPG or iGluRs antagonists. Glutamate suppressed substance P- and 5-HT-evoked slow EPSP-like responses and potentiated norepinephrine-induced slow IPSP-like responses. The results suggest that group I mGluRs mediate glutamate-induced suppression of slow EPSPs and potentiation of slow IPSPs in S-type uniaxonal submucosal neurons.

Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2.

1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Actions of bradykinin on electrical and synaptic behavior in the myenteric and submucous plexuses of guinea-pig small intestine

Actions of bradykinin on electrical and synaptic behavior in the myenteric and submucous plexuses of guinea-pig small intestine

Suppression of cAMP formation by adenosine in myenteric ganglia from guinea-pig small intestine

Effects of the adenosine receptor agonist 2-chloro- N 6-cyclopentyl-adenosine (CCPA) on stimulation of cAMP formation by histamine, 5-hydroxytryptamine, substance P and forskolin were determined for enzymatically dissociated ganglia from the myenteric plexus of guinea-pig small intestine. Each of the 4 substances stimulated cAMP production. CCPA blocked the stimulation of cAMP by histamine, but not by 5-hydroxytryptamine or substance P. CCPA marginally suppressed stimulation by forskolin. CCPA alone suppressed basal levels of cAMP. The adenosine receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) reversed the inhibitory action of CCPA on stimulation of cAMP formation by histamine. Exposure to adenosine deaminase or CPT increased cAMP in the ganglia. The results are consistent with a hypothesis that stimulation of adenylate cyclase and elevation of intraneuronal cAMP in enteric neurons are steps in the signal transduction cascade for the excitatory actions of 5-hydroxytryptamine, substance P and histamine. They are consistent also with an original hypothesis from electrophysiologic studies which states that stimulation of adenosine A 1 receptors suppresses cAMP formation and thereby slow synaptic excitation in response to histamine, but not to 5-hydroxytryptamine or substance P. The results support evidence from intracellular microelectrode studies which suggested that endogenous adenosine accumulates to levels sufficient for tonic suppression of cAMP formation in myenteric ganglia in vitro.

Actions of tumor necrosis factor (TNFα) on electrical and synaptic behavior in the submucous plexus of guinea-pig small intestine: [formula omitted], D.H. Zafirov, H.J. Cooke and J.D. Wood, Dept. of Physiol., The Ohio State Univ. Col. of Med., Columbus, OH

Actions of tumor necrosis factor (TNFα) on electrical and synaptic behavior in the submucous plexus of guinea-pig small intestine: [formula omitted], D.H. Zafirov, H.J. Cooke and J.D. Wood, Dept. of Physiol., The Ohio State Univ. Col. of Med., Columbus, OH

Identification of tachykinin receptors on neurons identified morphologically in the myenteric and submucous plexuses of guinea-pig small intestine: [formula omitted], D.H. Zafirov, P. Mohanraj, H.J. Cooke and J.D. Wood. Dept. of Physiol., The Ohio State Univ. Col. Med., Columbus, OH

Identification of tachykinin receptors on neurons identified morphologically in the myenteric and submucous plexuses of guinea-pig small intestine: [formula omitted], D.H. Zafirov, P. Mohanraj, H.J. Cooke and J.D. Wood. Dept. of Physiol., The Ohio State Univ. Col. Med., Columbus, OH

Determination of levels of cyclic AMP in the myenteric plexus of guinea-pig small intestine

Enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine were used to investigate changes in levels of cyclic 3',5'-adenosine monophosphate (cAMP) in response to stimulation of adenylate cyclase by forskolin and inhibition of phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX). A linear relation with a positive correlation coefficient greater than 0.98 was found between: (1) amount of cAMP and number of ganglia; (2) amount of protein and number of ganglia; (3) amount of DNA and amount of protein; (4) amount of DNA and number of ganglia. Basal levels of cAMP were 2.25 +/- 0.21 fmol per ganglion for 900 ganglia. Forskolin stimulated a dose-dependent increase in cAMP over a concentration range of 0.05 to 50 microM, with a level of 18.6 +/- 4.9 fmol/ganglion at 50 microM forskolin. The inactive forskolin analog 1,9-dideoxyforskolin did not elevate cAMP. Addition of IBMX to the incubation medium stimulated a dose-dependent increase in cAMP over a concentration range of 0.1-1000 microM, with a level of 17.58 +/- 3.38 fmol/ganglion at 1000 microM IBMX. Application of 1 mM IBMX strongly potentiated the stimulating action of forskolin on cAMP levels. Our results derived from direct determination of cAMP changes in small intestinal myenteric ganglia are consistent with existing electrophysiological evidence for second messenger function of cAMP in slow synaptic modulation of excitability in AH/Type 2 neurons of the enteric nervous system.

Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine

It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.

Calcitonin gene-related peptide: A potent new peptidergic messenger substance in the myenteric plexus of guinea-pig small intestine

Calcitonin gene-related peptide: A potent new peptidergic messenger substance in the myenteric plexus of guinea-pig small intestine