Purinergic Stimulation Research Articles

Intracellular calcium events activated by ATP in murine colonic myocytes.

ATP is a candidate enteric inhibitory neurotransmitter in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca(2+)-activated K(+) (SK) channels. Coupling between ATP stimulation and SK channels may be mediated by localized Ca(2+) release. Isolated myocytes of the murine colon produced spontaneous, localized Ca(2+) release events. These events corresponded to spontaneous transient outward currents (STOCs) consisting of charybdotoxin (ChTX)-sensitive and -insensitive events. ChTX-insensitive STOCs were inhibited by apamin. Localized Ca(2+) transients were not blocked by ryanodine, but these events were reduced in magnitude and frequency by xestospongin C (Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we have termed the localized Ca(2+) events in colonic myocytes "Ca(2+) puffs. " The P(2Y) receptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of Ca(2+) puffs. 2-MeS-ATP also increased STOCs in association with the increase in Ca(2+) puffs. Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, a P(2) receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca(2+) transients and the effects of 2-MeS-ATP on Ca(2+) puffs and STOCs were blocked by U-73122, an inhibitor of phospholipase C. Xe-C and ryanodine also blocked responses to 2-MeS-ATP, suggesting that, in addition to release from IP(3) receptor-operated stores, ryanodine receptors may be recruited during agonist stimulation to amplify release of Ca(2+). These data suggest that localized Ca(2+) release modulates Ca(2+)-dependent ionic conductances in the plasma membrane. Localized Ca(2+) release may contribute to the electrical responses resulting from purinergic stimulation.

Antagonists and the purinergic nerve hypothesis: 2,2′-pyridylisatogen tosylate (PIT), an allosteric modulator of P2Y receptors. A retrospective on a quarter century of progress

2,2′-Pyridylisatogen tosylate (PIT) is a selective antagonist of P2Y responses in smooth muscle and does not antagonise the effects of adenosine. Responses to purinergic nerve stimulation are resistant to PIT. PIT is an allosteric modulator of responses to ATP in recombinant P2Y 1 receptors expressed in Xenopus oocytes with potentiation of ATP at low concentrations (0.1–10 μM) and antagonism at higher ones (>10 μM). A radioligand binding profile showed that PIT did not interact with any other receptors, with the exception of low affinity for the adenosine A 1 receptor (p K i, 5.3). The compound recognises purine sites and then may cause irreversible binding to sulfhydryl groups following prolonged incubation or high concentrations. PIT is a potent spin trapper.

Phospholipase A2 and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells

The signal transduction involved in the purinergic stimuli-induced activation of protein kinase C (PKC) in CHO-K1 cells was investigated. Purinergic stimuli such as adenosine triphosphate and uridine triphosphate induced a transient translocation of PKC epsilon, gamma, and delta from the cytoplasm to the plasma membrane. These translocations were blocked by an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), but not by an inhibitor of phosphatidylcholine-specific PLC. A diacylglycerol (DAG) analogue also induced reversible translocations of PKC gamma, epsilon, and delta from the cytoplasm to the plasma membrane, while the calcium ionophore A23187 caused a similar translocation of only the gamma subtype. These results confirm that the hydrolysis of phosphatidylinositol-2-phosphate by PLC and the subsequent generation of DAG and increase in Ca(2+ )are involved in the purinergic stimuli-induced translocation of PKC. A DAG antagonist, 1-o-hexadecyl-2-o-acetyl-glycerol, blocked the DAG analogue-induced translocations of all PKC subtypes tested but failed to inhibit the purinergic stimuli-induced translocations of PKC epsilon and gamma. The DAG antagonist could not block the ATP- and UTP-induced translocation of PKC epsilon even in the absence of extracellular Ca(2+). Co-application of the DAG antagonist and a phospholipase A(2) (PLA(2)) inhibitor such as aristolochic acid, arachidonyltrifluoromethyl ketone, or bromoenol lactone inhibited the purinergic receptor-mediated translocation of PKC epsilon although each PLA(2) inhibitor alone did not block the translocation. In contrast to the epsilon subtype, ATP-induced translocation of PKC gamma was observed in the presence of both the PLA(2) inhibitor and the DAG antagonist. However, it is noteworthy that re-translocation of PKC gamma was hastened by the PLA(2) inhibitor. Furthermore products of PLA(2), such as lysophospholipids and fatty acids, induced the translocation of PKC gamma and epsilon in a dose dependent manner, but not delta. These results indicate that, in addition to PLC and DAG, PLA(2) and its products are involved in the purinergic stimuli-induced translocation of PKC epsilon and gamma in CHO-K1 cells. Each subtype of PKC in CHO-K1 cell is individually activated in response to a purinergic stimulation.

Enhanced negative chronotropy by inhibitory receptors in transgenic heart overexpressing β 2-adrenoceptors

Transgenic (TG) mice overexpressing β 2-adrenoceptors (AR) in the heart have enhanced β-adrenergic activity. Since the degree of β-adrenergic activation influences the negative chronotropic control of heart rate (HR), we studied the inhibitory effect of cholinergic and purinergic stimulation on HR in TG and wild-type (WT) control mice. Bradycardia in response to vagal nerve stimulation and administration of acetylcholine or adenosine was studied in anesthetised animals and perfused hearts. Basal HR was significantly higher in TG than WT mice ( P<0.01). Electrical stimulation of vagal nerves (1–32 Hz) induced a Hz-dependent reduction in HR and the response was more pronounced in TG than WT groups ( P<0.01). In perfused hearts, HR reduction by acetylcholine (ACh) was more pronounced with EC 50 110-fold lower in TG than WT hearts. Adenosine-induced bradycardia, which was abolished by a P 1 antagonist, was more pronounced in TG hearts. After pre-treatment with pertussis toxin (PT, 100 μg/kg), bradycardia by vagal nerve stimulation or ACh remained unchanged in WT, but markedly inhibited in TG hearts (both P<0.01). Conversely, inhibiting guanylyl cyclase with LY83583 (30 μM) or nitric oxide synthase with l-NMMA (100 μM) attenuated HR reduction by vagal nerve stimulation in WT but not in TG hearts. Immunobloting assay showed similar G iα2 abundance in TG and WT hearts. Thus, cardiac overexpression of β 2AR with high β-adrenergic activity leads to hypersensitivity of inhibitory receptors controlling HR due to increase in activity of PT-sensitive G-proteins.

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes.

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to -50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca(2+) with equimolar Mn(2+) caused STOCs to "run down. " Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs" remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca(2+)-activated K(+) channels (SK channels). Application of ATP or 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, a P(2) receptor blocker. Similarly, pretreatment of cells with U-73122 (1 microM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P(2Y) receptors results in localized Ca(2+) release via PLC- and IP(3)-dependent mechanisms. Release of Ca(2+) is coupled to STOCs, which are composed of currents mediated by large-conductance Ca(2+)-activated K(+) channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

Purinergic stimulation of ciliary activity

The mucociliary system is responsible for maintaining the airways clean of inhaled particles and pathogens. This task is performed by ciliated cells that transport the mucus from the lungs to the upper airways. The energy transferred by the cilium to the mucus blanket is proportional to the square of the ciliary beat frequency (CBF), hence CBF has a profound effect on the efficiency of mucociliary clearance and impairment of ciliary activity may lead to severe pathological conditions by jeopardizing mucociliary clearance. Extracellular ATP is one of the most potent activators of ciliary beating. It acts through purinergic receptors located on the cell surface. Ciliary stimulation is achieved by means of elevation in intracellular calcium concentration and activation of protein kinases. This article outlines the mechanism of purinergic ciliary stimulation revealed to date.

Perivascular Purinergic Nerve-Induced Vasoconstrictions in Canine Isolated Splenic Arteries

We tried to induce selective perivascular purinergic nerve stimulation in isolated canine splenic arterial preparations, using the cannula insertion method. Under the conditions of periarterial electrical stimulation (ES), i.e., trains of 1, 3 and 10 pulses, 1-ms pulse duration and 10-V amplitude at 1 Hz, monophasic vasoconstriction was consistently induced. The ES-induced vasoconstriction was not influenced by prazosin in doses that completely inhibited noradrenaline-induced vasoconstrictions, but it was suppressed by alpha,beta-methylene ATP, a P2X purinoceptor desensitizer. Thus, it is indicated that a selective purinergic transmitter release is readily obtained in the isolated splenic arterial preparation.

Pharmacological analysis of vasoconstrictor responses to periarterial purinergic nerve stimulation.

1. Periarterial electrical nerve stimulation at a low frequency (1 Hz) readily induced a vasoconstrictor response of the canine splenic artery in a pulse number-related manner (1-30 pulses of trains). The vasoconstrictor response to trains of up to 10 pulses at 1 Hz of stimulation appeared to be monophasic, whereas it became clearly distinguished into two phases at a longer train of 30 pulses. 2. The monophasic vasoconstrictor responses to trains of 1, 3 or 10 pulses were not modified by an alpha1-adrenoceptor blocking agent, prazosin (0.1 microM), but were completely inhibited by the P2X receptor desensitization with alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-methylene ATP; 1 microM). The 1st phase of vasoconstriction induced by a train length of 30 pulses was not influenced by the treatment with prazosin, but was abolished by alpha,beta-methylene ATP. The 2nd phase response was markedly inhibited by prazosin, and the remaining response of this phase was blocked by alpha,beta-methylene ATP. 3. Rauwolscine (0.3 microM), an alpha2-adrenoceptor antagonist, enhanced the vasoconstrictor responses to trains of 1, 3 or 10 pulses. Particularly at 10 pulses of electrical stimulation, the vasoconstrictor responses were significantly potentiated. The blockade of neuronal uptake of noradrenaline with imipramine (1 microM) did not affect the vasoconstrictor responses to trains of 1, 3 or 10 pulses. 4. It is concluded that short pulse trains of stimulation at a low frequency may selectively activate a purinergic component of sympathetic cotransmission, and the prejunctional alpha2-adrenergic feedback mechanism may tonically participate into the modulation of ATP release. Imipramine-sensitive neuronal uptake mechanism may not play an important role in regulating vascular responses to periarterial purinergic nerve stimulation.

Muscarinic receptor heterogeneity in follicle-enclosed Xenopus oocytes.

1. Ionic current responses elicited by acetylcholine (ACh) in follicle-enclosed Xenopus oocytes (follicles) were studied using the two-electrode voltage-clamp technique. ACh generated a fast chloride current (Fin) and inhibited K+ currents gated by cAMP (IK,cAMP) following receptor activation by adenosine, follicle-stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs. 2. Inhibition of IK,cAMP was about 100 times more sensitive to ACh than Fin activation; the half-maximal effective concentrations (EC50) were 6.6 +/- 0.4 and 784 +/- 4 nM, respectively. 3. Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 +/- 0.07 nM, whilst the IC50 for IK,cAMP inhibition was 5.9 +/- 0.2 microM. 4. Oxotremorine, a muscarinic agonist, preferentially stimulated IK, cAMP inhibition (EC50 = 15.8 +/- 1.4 microM), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 +/- 0.7 microM. 5. Fin elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell-oocyte coupling. 6. Using reverse transcription-PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in IK,cAMP inhibition. 7. In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype and the other the M3 subtype. These receptors are coupled to distinct membrane mechanisms leading to independent regulation of two membrane conductances.

Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore.

The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X(7) ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X(7) receptors or 2) MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-gamma, a maneuver known to upregulate P2Z/P2X(7) receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X(7) receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X(7) channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)); the initial increase reflects MTX-induced Ca(2+) influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca(2+)](i) and ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X(7) receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X(7) receptor, MTX-induced increases in [Ca(2+)](i) and ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca(2+)](i) had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22 degrees C, a characteristic shared by the P2Z/P2X(7)-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X(7) receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

Prostacyclin enhances stretch-induced surfactant secretion in alveolar epithelial type II cells.

Inhalative vasodilator therapy, employing gaseous nitric oxide (NO) or aerosolized prostaglandin PGI(2), is of interest for regional pulmonary vasodilation in ARDS and pulmonary hypertension. We investigated the impact of the NO donor spermine NONOate as well as PGI(2) and its stable chemical analog iloprost on cultured rat alveolar epithelial type II cell (ATII) surfactant secretion. The NO donor provoked a significant increase in the ATII cGMP content, further enhanced by type V phosphodiesterase (PDE) inhibition, but affected neither baseline nor mechanical stretch-induced surfactant secretion. The prostanoids caused a marked increase in the epithelial cAMP content, further amplified by coadministration of type III/IV PDE inhibitors. Baseline surfactant secretion was not altered by this approach, but mechanical stretch-induced liberation of surfactant was significantly increased, most prominently in the ATII with the highest cAMP levels due to the presence of both iloprost and PDE III/IV inhibitors. In contrast, epithelial phosphoinositide metabolism, well responsive to purinergic stimulation as positive control, was unchanged in prostanoid-exposed cells. We conclude that the PGI(2)-cAMP axis, but not the NO-cGMP axis, forwards a markedly enhanced secretory response to the physiological stimulus of cell surface stretching, which may be relevant for therapeutic use of these agents.

Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea.

1. Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation. 2. Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin. 3. The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3', 5'-cyclic monophosphothioate triethylamine (Rp-cAMPS). 4. Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i. 5. Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin. 6. The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP.

ATP Can Stimulate Exocytosis in Rat Brown Adipocytes without Apparent Increases in Cytosolic Ca 2+ or G Protein Activation

Extracellular ATP activates large increases in cell surface area and membrane turnover in rat brown adipocytes (Pappone, P. A., and Lee, S. C. 1996. J. Gen. Physiol. 108:393–404). We used whole-cell patch clamp membrane capacitance measurements of membrane surface area concurrently with fura-2 ratio imaging of intracellular calcium to test whether these purinergic membrane responses are triggered by cytosolic calcium increases or G protein activation. Increasing cytosolic calcium with adrenergic stimulation, calcium ionophore, or calcium-containing pipette solutions did not cause exocytosis. Extracellular ATP increased membrane capacitance in the absence of extracellular calcium with internal calcium strongly buffered to near resting levels. Purinergic stimulation still activated exocytosis and endocytosis in the complete absence of intracellular and extracellular free calcium, but endocytosis predominated. Modulators of G protein function neither triggered nor inhibited the initial ATP-elicited capacitance changes, but GTP γS or cytosolic nucleotide depletion did reduce the cells’ capacity to mount multiple purinergic responses. These results suggest that calcium modulates purinergically-stimulated membrane trafficking in brown adipocytes, but that ATP responses are initiated by some other signal that remains to be identified.

P2 receptor modulation of voltage-gated potassium currents in Brown adipocytes.

Using patch voltage-clamp techniques, we find there are two components to the voltage-gated potassium current (IKv) in rat brown adipocytes. The components differ in their gating and responses to purinergic stimulation, but not their pharmacology. IKv-A recovers from inactivation at physiological membrane potentials, while IKv-B inactivation recovers at more negative potentials. Both currents are >90% blocked by similar concentrations of quinine and tetraethylammonium, but not by beta-dendrotoxin, charybdotoxin, or apamin. The two current components are differentially modulated by extracellular ATP. ATP shifts the voltage dependence of IKv-A inactivation negative by 38 +/- 5 mV (n = 35, +/-SEM) and shifts activation by -14 +/- 2 mV in whole-cell experiments. ATP did not affect the steady state inactivation voltage dependence of IKv-B, but did apparently convert IKv-A into IKv-B. The pharmacology of the inactivation shift is consistent with mediation by a P2 purinergic receptor. Purinergic stimulation of perforated-patch clamped cells causes hyperpolarizing shifts in the window current of IKv-A by shifting inactivation -18 +/- 4 mV and activation -7 +/- 2 mV (n = 16). Since perforated-patch recordings will most closely resemble in vivo cell responses, this ATP-induced shift in the window current may facilitate IKv activation when the cell depolarizes. IKv activity is necessary for the proliferation and differentiation of brown adipocytes in culture (Pappone, P.A., and S.I. Ortiz-Miranda. 1993. Am. J. Physiol. 264:C1014-C1019) so purinergic modulation of IKv may be important in altering adipocyte growth and development.

Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1.

The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.

EFFECTS OF ATROPINE, ISOPROTERENOL AND PROPRANOLOL ON THE RABBIT BLADDER CONTRACTION INDUCED BY INTRA-ARTERIAL ADMINISTRATION OF ACETYLCHOLINE AND ATP

Introduction In the rabbit, both cholinergic and purinergic nerves mediate bladder contraction. Acetylcholine is the neurohumoral transmitter for the cholinergic nerves, and ATP is the neurohumoral transmitter for purinergic innervation. Beta-adrenergic stimulation mediates relaxation of the bladder. In the current study, we investigated the effects of atropine, isoproterenol and propranolol on the bladder contraction induced by intra-arterial administration of acetylcholine and ATP. Methods Mature male New Zealand White rabbits were used in this study. A polyethylene catheter with an outer diameter of 0.043 inches was inserted through the rabbit's right femoral artery until it reached the lower abdominal aorta. An 8 F catheter was inserted through the urethral orifice into the bladder and secured by tying a 2-0 silk ligature around the bladder neck. The catheter was connected to an infusion pump and a pressure transducer by a 3-way valve. After 15 ml. of saline was infused into the bladder, an intra-arterial administration of acetylcholine and ATP was infused and the change of intravesical pressure was quantitated and recorded with a Grass model 7D polygraph. The procedure was repeated after a 5-minute pretreatment with atropine, isoproterenol or propranolol. Results The results are summarized as follows: 1) Baseline intravesical pressure was not altered by pretreatment with atropine. Pretreatment with atropine shifted the dose-response curve of acetylcholine to the right and the maximal response was reduced by 9%, 49% and 77% respectively with pretreatment with atropine 10 −8, 10 −7 and 10 −6 mole/kg. The dose-response curve of ATP was not significantly affected by pretreatment with atropine. 2) Baseline intravesical pressure was lowered by pretreatment with isoproterenol. Pretreatment with isoproterenol shifted both dose-response curves of acetylcholine and ATP rightward. The maximal response of acetylcholine was reduced by 10%, 26% and 37% respectively, and the maximal response of ATP was reduced by 6%, 31% and 43% respectively by pretreatment with isoproterenol 10 −8, 10 −7 and 10 −6 mole/kg. 3) Baseline intravesical pressure was not changed by pretreatment with propranolol. Both dose-response curves of acetylcholine and ATP were not significantly affected by pretreatment with propranolol. Summary In conclusion, pretreatment with atropine inhibited acetylcholine-induced bladder contraction, but had no effect on ATP-induced contraction. Pretreatment with isoproterenol significantly inhibited both contractile stimulation by acetylcholine and ATP. Pretreatment with beta-adrenergic antagonist had no effect on the bladder contraction induced either by acetylcholine or by ATP. Thus, although beta-adrenergic stimulation is capable of significantly inhibiting the contractile responses to both cholinergic and purinergic stimulation, under normal conditions, sympathetic nerves do not modulate either cholinergic or purinergic stimulation.

Purinergic activation of a tyrosine kinase-dependent pathway in cardiac cells

This overview focuses on the role of cytosoluble tyrosine kinases in the purinergic regulation of cardiac function. Cardiac cells express many cytosolic tyrosine kinases, including pp60c-src, p59c-fyn, Csk, FAK, and Tec. Purinergic stimulation of cardiomyocytes increases the activity of pp60c-src and p59c-fyn and induces phosphorylation of FAK. This signaling pathway leads to phosphorylation of many proteins, including PLCγ, the major PLC isoform in heart, and AE1, the predominant cardiac Cl/HCO3 exchanger. Tyrosine kinase-mediated phosphorylation of PLCγ and AE1 allows the cardiomyocyte to regulate both its Ca2+ and H+ homeostasis, respectively. The existence of other cardiac intracellular substrates of tyrosine kinases, targets of the purinergic stimulation as well as the physiological relevance of this signaling pathway, is discussed. Drug Dev. Res. 45:427–433, 1998. © 1998 Wiley-Liss, Inc.

Cytoskeletal Assembly and ATP Release Regulate Astrocytic Calcium Signaling

We have studied the role of actin fiber assembly on calcium signaling in astrocytes. We found that (1) after astrocytes have been placed in culture, it takes several hours for organization of the definitive actin cytoskeleton. Actin organization and the number of cells engaged in calcium signaling increased in parallel. (2) Disruption of the actin cytoskeleton attenuated the calcium wave propagation; cytochalasin D treatment reduced the number of astrocytes engaged in calcium signaling. (3) Propagation of calcium waves depends on cytoskeletal function; inhibition of myosin light chain kinase suppressed wave activity. (4) Astrocytic calcium signaling is mediated by release of ATP and purinergic receptor stimulation, because agents that interfere with this cascade attenuated or reduced calcium signaling. Because purinergic receptors are fully functional shortly after plating and not affected by cytochalasin D, these observations indicate that cytoskeleton organization is a prerequisite for interastrocytic calcium signaling mediated by release of ATP.

Differences in the sensitivity to purinergic stimulation of myelinating and non-myelinating Schwann cells in peripheral human and rat nerve.

Schwann cells of the peripheral nervous system are distinguished by morphological and functional criteria in myelinating and non-myelinating subtypes. We and others have previously reported that Schwann cells in isolated peripheral human and rat nerve respond to extracellular application of ATP with a rise in the intracellular free calcium concentration [Ca2+]i. In the present study, the receptors mediating these Ca2+ transients have been investigated in myelinating and non-myelinating Schwann cells of intact fascicles of isolated human sural nerves, rat ventral roots, and rat vagus nerves. Microfluorometry and confocal laser scanning was used on preparations stained with the Ca2+-sensitive dyes Calcium Green-1 and Fura Red. In myelinating Schwann cells of human and rat nerves, the ATP-induced rise of [Ca2+]i resulted from the activation of a P2Y2 purinoceptor subtype (rank order of potency: UTP > or = ATP >> 2-MeSATP = ADP). In contrast, in non-myelinating Schwann cells, Ca2+ transients were produced by activation of a P2Y1 purinoceptor subtype (rank order of potency: 2-MeSATP > ATP > ADP >> UTP). The P1 agonist adenosine and alpha,alpha-meATP did not evoke Ca2+ signals. Ca2+ transients in both types of Schwann cells were found to be due to Ca2+ release from cyclopiazonic acid-sensitive intracellular stores. However, inhibition by suramin was only found in non-myelinating Schwann cells. These findings indicate that mammalian Schwann cells express phenotype-specific P2Y receptor subtypes.

Specific activation of adenylyl cyclase V by a purinergic agonist

The present study was designed to investigate whether and how the purinergic stimulation of rat ventricular myocytes modulates the cAMP-dependent pathway. Stimulation of cardiomyocytes with ATPγS in the presence of the phosphodiesterase inhibitor IBMX increases by 3-fold intracellular cAMP content. In contrast to β-adrenergic stimulation, the purinergic stimulation of adenylyl cyclase was not inhibited by activation or enhanced by inhibition of a G i protein. Forskolin did not potentiate the effect of extracellular ATPγS on intracellular cAMP content but the effect of isoproterenol did. Like isoproterenol, the purinergic agonist decreased subsequent ADP-ribosylation of a 45 kDa G αs by cholera toxin. ATPγS also increased cAMP content in neonatal rat cardiomyocytes, a cell type that expresses a long form of G s protein (α s, 52 kDa) in contrast to adult rat cardiomyocytes that express mostly a short form of G s protein (α s, 45 kDa). Both purinergic and β-adrenergic agonists increased cAMP in HEK 293 cells expressing type V adenylyl cyclase while cAMP was only increased by β-adrenergic stimulation of HEK expressing type IV or VI adenylyl cyclases. Thus, we propose that the purinergic and β-adrenergic stimulations differentially activate adenylyl cyclase isoforms in rat cardiomyocytes and that adenylyl cyclase V is the specific target of the purinergic stimulation.