Neuroligands evoke calcium signaling in cultured myenteric neurons.

BACKGROUND: Myenteric neurons, which control gut motility and absorption, are heterogeneous in structure, electrophysiology, and neuropeptide content. We hypothesized that myenteric neurons would display heterogeneous responses to neuroligands found in enteric tissue. We examined increases in intracellular calcium ([Ca2+]i), an important second messenger, evoked by selected neuroligands in cultured myenteric neurons. METHODS: Primary cultures of guinea pig myenteric neurons were characterized by using a standard immunocytochemical stain for microtubule-associated protein (MAP2). Increases in [Ca2+]i were measured by digital imaging microscopy. Results expressed as mean +/- SEM, Student's t test. RESULTS: Cultured myenteric neurons examined with phase contrast and Nomarsky optics extend processes and stain positively with the neuron-selective stain MAP2. Histamine did not evoke calcium mobilization in cultured neurons, although progressive increases in [Ca2+]i were seen with bradykinin, glutamate, serotonin, cholecystokinin, bombesin, adenosine triphosphate (ATP), substance P, and acetylcholine. Enteric neurons differed from one another in the ability to respond to one, two, or three of the agonist combinations tested. Time in culture did not alter the percentage of neurons responding to ATP, substance P, or acetylcholine. ATP evoked equivalent [Ca2+]i increases in neurons examined at the three time points, whereas significant decrements in neuronal calcium mobilization were seen after 8 days in culture with substance P or acetylcholine (p < 0.05 versus 1 day in vitro). CONCLUSIONS: Cultured myenteric neurons extend processes and retain expression of neuron-specific antigens. Selected neuroligands found in enteric tissue increase [Ca2+]i, as a second messenger in subsets of myenteric neurons. Heterogeneity exists among cultured neurons in their ability to respond to ligand combinations. Changes in [Ca2+]i induced by neuroligands in myenteric neurons may be altered with time in vitro.

Pituitary adenylate cyclase-activating peptide stimulates amylase release and cyclic adenosine monophosphate production in pancreatic acinar cells.

BACKGROUND: Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide member of the secretin/glucagon family of peptides, which also includes vasoactive intestinal peptide (VIP). This study was designed to examine the effects of PACAP-38 on pancreatic exocrine function in vitro. METHODS: Amylase release and signal transduction pathways were examined by using dispersed guinea pig acinar cells. RESULTS: PACAP-38 produced dose-dependent increases in amylase release. Coincubation of PACAP-38 (1 nmol/L) additively augmented amylase release stimulated by cholecystokinin, carbachol, or bombesin. Coexposure with PACAP-38 did not affect amylase release in response to maximally stimulatory concentrations of VIP (1 nmol/L). The VIP antagonist, [N-Ac-Tyr1,D-Phe2]-GRF(1-29)-NH2, abolished amylase release in response to either PACAP-38 or VIP. Dose-dependent increases in cyclic adenosine monophosphate production were noted on exposure to PACAP-38, with a 70-fold increment relative to the control value at 1 nmol/L PACAP-38. Inositol phosphate turnover and intracellular Ca2+ levels were not affected by PACAP-38 exposure. CONCLUSIONS: In guinea pig pancreatic acini, VIP preferring receptors for PACAP-38 are functionally linked to adenylate cyclase.

Bombesin-mediated calcium fluxes in myenteric plexus neurons.

Dual excitation microfluorimetry (Fura-2) was used to measure changes in intracellular calcium ([Ca2+]i) in individual cultured guinea pig myenteric neurons. Bombesin (5-500 nM) induced concentration-dependent increases in [Ca2+]i responses, with a maximal effect at 500 nM (56% of neurons responding, mean peak Ca2+ response 244 +/- 25 nM vs. basal 65 +/- 7 nM). Removal of Ca2+ from the median did not affect the initial [Ca2+]i peak but eliminated the subsequent plateau phase. The [Ca2+]i responses to bombesin was abolished by preincubation with thapsigargin (1 microM), a Ca(2+)-ATPase inhibitor (91 +/- 7% inhibition). [Ca2+]i responses to bombesin were inhibited by U73122 (1 microM), an inhibitor of phospholipase C (84 +/- 6% inhibition).

Tachykinin neuropeptide-evoked intracellular calcium transients in cultured guinea pig myenteric neurons.

Substance P and related tachykinins are present in the mammalian gut and act as neurotransmitters. Microfluorimetric measurement of intracellular calcium ([Ca2+]i) was used to study tachykinin-sensitive myenteric neurons. Substance P (0.001-10 microM) evoked concentration-dependent increases in percentage of neurons responding (6-75%) and delta [Ca2+]i (88 +/- 24 to 212 +/- 16 nM). Neurokinin A (0.001-1 microM) produced similar responses. Removal of extracellular Ca2+ abolished substance P-induced Ca2+ signals, as did the addition of the Ca2+ channel blockers lanthanum chloride (5 mM) and nickel chloride (2.5 mM). Both nifedipine (1-50 microM) and diltiazem (1-50 microM) inhibited substance P-evoked Ca2+ responses in a dose-dependent manner. Substance P and related tachykinins evoke Ca2+ signaling in cultured myenteric neurons by the influx of extracellular Ca2+ through L and N-type plasma membrane Ca2+ channels.

Acetylcholine-induced calcium signaling associated with muscarinic receptor activation in cultured myenteric neurons.

BACKGROUND: Within the enteric nervous system, acetylcholine (ACh) is an important neurotransmitter. Experimental evidence has suggested that in myenteric neurons, calcium plays a key role in the coupling of cholinergic receptors to secretory responses. STUDY DESIGN: We investigated the effects of ACh on intracellular calcium concentration ([Ca2+]i) in individual myenteric neurons using fura-2 microspectrofluorometry. RESULTS: Resting [Ca2+]i in myenteric neurons was 62.5 +/- 3 nM. Acetylcholine produced dose-dependent increases in [Ca2+]i in myenteric neurons. As the concentration of ACh was increased from 0.1 to 100 microM, both the peak [Ca2+]i response as well as the percentage of responding neurons progressively increased, with a maximal effect at 100 microM (347 +/- 31 nM, 95 percent of neurons). The effect of ACh was not sensitive to pertussis toxin (100 ng/mL). Calcium ion (Ca2+) responses to ACh were abolished by removal of extracellular Ca2+ as well as exposure to nifedipine (10 microM). Characterization of the specific muscarinic subtype(s) involved in ACh-mediated Ca2+ transients was performed using the specific antagonists pirenzepine (M1), gallamine (M2), and 4-DAMP (M3). Pirenzepine (1 microM) blocked increases in [Ca2+]i induced by ACh; gallamine (1 microM) and 4-DAMP (1 microM) had no significant effect. Intracellular Ca2+ responses to ACh were not affected by incubation with the phorbol ester tetradecanoylphorbol-13-acetate (1 microM). CONCLUSIONS: These findings suggest that ACh induces increases in [Ca2+]i in myenteric neurons by promoting influx of extracellular Ca2+ through L-type voltage-dependent Ca2+ channels by activation of the M1 muscarinic receptor subtype. The Ca2+ response does not appear to involve a pertussis toxin-sensitive G protein.

Enteric glia exhibit P2U receptors that increase cytosolic calcium by a phospholipase C-dependent mechanism.

Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 mM), glutamate (100 microM), norepinephrine (10 microM), and substance P (1 microM) did not increase the intracellular calcium concentration ([Ca2+]i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 microM), bradykinin (11%; 10 microM), and histamine (31%; 100 microM), whereas 100% of glia responded to ATP (100 microM). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca2+]i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca2+]i evoked, ATP = UTP > ADP > beta, gamma-methyleneadenosine 5'-triphosphate >> 2-methylthioadenosine 5'-triphosphate = alpha,beta-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P2U receptor. Depletion of D-myo-inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 microM) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 microM), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca2+]i. Our data suggest that glial responses to ATP are mediated by a P2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.

Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism.

In the myenteric plexus, ATP is released as a neurotransmitter by "purinergic" nerves, relaxing visceral smooth muscle. We report a signal transduction mechanism for ATP in cultured myenteric neurons involving receptor-mediated release of intracellular Ca2+ stores. Primary cultures of myenteric neurons from guinea pigs taenia coli were loaded with the Ca2+ indicator fura 2-acetoxymethyl ester (AM) and examined using digital imaging microscopy. Superfusion of single neurons with ATP (0.01-1,000 microM) resulted in concentration-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) that were independent of extracellular Ca2+. Decrements in peak [Ca2+]i were seen with repetitive ATP exposure. Responsiveness of myenteric neurons to purinergic agonists (100 microM) was consistent with action at a neuronal P 2y purinoceptor: 2-chloro-ATP = ATP = 2-methyl-thio-ATP (MeSATP) > ADP > alpha, beta-MeATP = beta,gamma-MeATP > AMP > adenosine. ATP-evoked Ca2+ transients were inhibited dose dependently by suramin, a nonspecific P2 antagonist, and reactive blue 2, a specific P 2y antagonist. ATP and cyclopiazonic acid (30 microM) appear to release an identical intracellular Ca2+ store. Preincubation with the aminosteroid U-73122 (10 microM) inhibited ATP-evoked Ca2+ transients by 71 +/- 7%, whereas phorbol ester pretreatment (phorbol 12-myristate 13-acetate, 100 nM, 5 min) caused a 76 +/- 4% inhibition. Peak [Ca2+]i evoked by ATP was not affected by preincubation with pertussis toxin (100 ng/ml, 24 h) or nifedipine (10 microM). These data suggest a signal transduction mechanism for ATP in cultured myenteric neurons involving purinoceptor-mediated activation of phospholipase C (PLC), with release of D-myo-inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores.

Caffeine- and ryanodine-sensitive Ca2+ stores in cultured guinea pig myenteric neurons.

In single fura 2-loaded myenteric neurons, caffeine caused concentration-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) that were quantal, saturable, and reversible. Inhibition of caffeine-induced Ca2+ release was demonstrated by ryanodine (1 microM), dantrolene (10 microM), and procaine (5 mM). Caffeine and cyclopiazonic acid (30 microM) released overlapping Ca2+ stores, whereas the caffeine-releasable pool was a subset of Ca2+ released by the Ca2+ ionophore ionomycin (4 microM). Both mild depolarization (7.5 mM KCl) and a submaximal concentration of caffeine (1 mM) produced neuronal [Ca2+]i oscillations in one-third of cells examined, which could be abolished by ryanodine (1 microM) or removal of extracellular Ca2+. Release of caffeine-sensitive Ca2+ stores induced influx of extracellular Ca2+. Immunolocalization using confocal microscopy revealed ryanodine receptor-like staining within the cytosol of cultured myenteric neurons.