Michael E. Bradley

Involvement of cyclic GMP in non‐adrenergic, non‐cholinergic inhibitory neurotransmission in dog proximal colon

1 Nitric oxide (NO) may serve as a non‐adrenergic, non‐cholinergic (NANC) neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. We tested whether guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) may serve as a second messenger in transducing the NO signal into inhibitory junction potentials (i.j.ps) and relaxation in the canine proximal colon. 2 The membrane permeable analogue of cyclic GMP, 8‐bromo cyclic GMP (8‐Br‐cyclic GMP) mimicked the effects of NO by hyperpolarizing cells near the myenteric border of the circular muscle layer and shortening slow waves in cells near the submucosal surface of the circular muscle layer. 8‐Br‐cGMP also inhibited spontaneous phasic contractions. 3 The specific cyclic GMP phosphodiesterase inhibitor, M&B 22948, hyperpolarized cells near the myenteric border and prolonged the duration of i.j.ps. M&B 22948 also inhibited phasic contractile activity. 4 Methylene blue failed to reduce significantly the amplitude and duration of i.j.ps and had variable effects on contractions. 5 Cyclic GMP levels were assayed in unstimulated muscles and in muscles exposed to exogenous NO and electrical field stimulation. Both stimuli hyperpolarized membrane potential, inhibited contractions, and elevated cyclic GMP levels. 6 Treatment of muscles with l‐NG‐nitroarginine methyl ester (l‐NAME) increased spontaneous contractile activity and lowered cyclic GMP levels. The inhibitory effect of M&B 22948 on contractions was greatly reduced after muscles were treated with l‐NAME. 7 These data support the concept that the effects of NANC nerve stimulation and NO (which may be one of the enteric inhibitory transmitters) may be mediated by cyclic GMP.

Evidence for a discrete UTP receptor in cardiac endothelial cells

1 We have examined the effects of various purine and pyrimidine nucleotides upon cells cultured from guinea‐pig cardiac endothelium (CEC), and find the P2Y‐agonist 2‐methylthioadenosine triphosphate (2MeSATP) to be a potent (EC50 = 85±10.2 nM) stimulator of increases in intracellular calcium concentrations, while uridine 5′‐triphosphate (UTP) and adenosine 5′‐triphosphate (ATP) are less potent but equipotent with one another (EC50s = 2.1±0.3 and 1.8±0.2 μm, respectively). 2 While the P2Y receptor exhibited rapid homologous desensitization, this had no effect upon subsequent responsiveness of CEC to either ATP or UTP. Effects of maximal concentrations of ATP and UTP were not only additive, but did not cross‐desensitize. Responses to UTP (but not to ATP or 2MeSATP) were blocked by treatment with pertussis toxin (PTX); all three nucleotides appeared to liberate calcium from an intracellular pool. 3 Suramin (30 μm) significantly (P<0.05) increased the EC50 for ATP‐dependent increases in intracellular calcium (5.3±2.2 μm vs. 2.0±0.9 μm in the absence of suramin), while it completely blocked the response to 2MeSATP. Suramin had no effect upon responses to UTP at concentrations of 100 μm. 4 We conclude that in addition to the P2Y and P2U subtypes of the ATP receptor, an additional receptor responsive to UTP but exhibiting no affinity for purine nucleotides is present in CEC; this ‘pyrimidine receptor’ liberates intracellular calcium via a G‐protein, and may partly mediate the contractile response to UTP in the coronary vasculature.

Cyclic GMP‐independent effects of nitric oxide on guinea‐pig uterine contractility

1 The role of nitric oxide (NO) in the regulation of uterine contractility has yet to be clearly defined. We evaluated the effect of NO (in the form of S‐nitroso‐L‐cysteine, CysNO) upon uterine contractility and guanosine 3′,5′‐cyclic monophosphate (cyclic GMP) accumulation in pregnant and nonpregnant guinea‐pig myometrium. 2 While CysNO had no effect upon spontaneous contractile activity in either pregnant or nonpregnant uterine tissues, addition of CysNO resulted in an immediate and reversible relaxation of oxytocin‐ or acetylcholine (ACh)‐evoked contractions. 3 Relaxation of agonist‐evoked contractions in response to CysNO was associated with significant elevations in intracellular cyclic GMP concentrations ([cyclic GMP]i). 4 Elevations in [cyclic GMP]i were not required for relaxation, as inhibition of guanylyl cyclase by methylene blue prevented [cyclic GMP]i accumulation while having no effect upon the ability of CysNO to relax agonist‐evoked contractions. 5 Addition of the cyclic GMP‐analogues, 8‐Br‐cyclic GMP and PET‐cyclic GMP, only at high concentrations, produced partial relaxation of agonist‐contracted tissues, suggesting the possibility that cyclic GMP may be sufficient but not necessary for myometrial relaxation. 6 Our studies not only provide evidence for a functional role for NO‐modulation of agonist‐evoked contractions in the pregnant and nonpregnant guinea‐pig uterus, but also that these occur by a mechanism which is not dependent upon guanylyl cyclase activity.

Chemotactic, mitogenic, and angiogenic actions of UTP on vascular endothelial cells.

Endothelial cells express receptors for ATP and UTP, and both UTP and ATP elicit endothelial release of vasoactive compounds such as prostacyclin and nitric oxide; however, the distinction between purine and pyrimidine nucleotide signaling is not known. We hypothesized that UTP plays a more important role in endothelial mitogenesis and chemotaxis than does ATP and that UTP is angiogenic. In cultured endothelial cells from guinea pig cardiac vasculature (CEC), both UTP and vascular endothelial growth factor (VEGF) were significant mitogenic and chemotactic factors; in contrast, ATP demonstrated no significant chemotaxis in CEC. In chick chorioallantoic membranes (CAM), UTP and VEGF treatments produced statistically significant increases in CAM vascularity compared with controls. These findings are the first evidence of chemotactic or angiogenic effects of pyrimidines; they suggest a role for pyrimidine nucleotides that is distinct from those assumed by purine nucleotides and provide for the possibility that UTP serves as an extracellular signal for processes such as endothelial repair and angiogenesis.

Nitric oxide regulation of monkey myometrial contractility

We evaluated the effect of the nitric oxide (NO) donor CysNO (S‐nitroso‐L‐cysteine) and endogenous NO upon spontaneous contractility in non‐pregnant cynomolgus monkeys. We also assessed the role of intracellular guanosine 3′,5′‐cyclic monophosphate ([cyclic GMP]i) as a second messenger for NO in monkey uterine smooth muscle. CysNO reduced spontaneous contractility by 84% (P<0.05) at maximal concentrations, and significantly elevated [cyclic GMP]i (P<0.05). However, increases in [cyclic GMP]i were not required for CysNO‐induced relaxations; CysNO inhibited contractile activity despite the complete inhibition of guanylyl cyclase by methylene blue or LY83,583. Analogues of cyclic GMP had no significant effect upon spontaneous contractile activity. L‐arginine produced a 62% reduction in spontaneous activity (P<0.05) while D‐arginine had no effect. The competitive nitric oxide synthase (NOS) inhibitor Nω‐nitro‐L‐arginine (L‐NOARG) not only blocked L‐arginine‐induced relaxations, but also significantly increased spontaneous contractile activity when added alone (P<0.05); the inactive D‐enantiomer of NOARG had no such effect. While both endogenous NO and the NO donor CysNO relax monkey myometrium, this effect is not causally related to CysNO‐induced elevations in [cyclic GMP]i. The failure of cyclic GMP analogues to alter monkey uterine smooth muscle tension also argues against a role for [cyclic GMP]i in the regulation of uterine contractility. Not only do these findings argue for the existence of a functionally‐relevant NOS in the monkey uterus, but increases in contractile activity seen in the presence of NOS inhibitors suggest a role for NO in the moment‐to‐moment regulation of contractile activity in this organ.

Molecular diversity of KVα- and β-subunit expression in canine gastrointestinal smooth muscles

Voltage-activated K+(KV) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the KV currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different KV channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of KV1.4, KV1.5, KV1.6, KV2.2, and KV4.3 transcripts in all regions of the GI tract examined. Transcripts encoding KV1.2, KVβ1.1, and KVβ1.2 subunits were differentially expressed. KV1.1, KV1.3, KV2.1, KV3.1, KV3.2, KV3.4, KV4.1, KV4.2, and KVβ2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these KV channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that KV1.2, KV1.4, KV1.5, and KV2.2 are expressed at the protein level in GI tissues and smooth muscle cells. KV2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of KV channel subunits.

Inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate binding sites in smooth muscle

1 We have previously demonstrated that activation of M3 muscarinic receptors increases inositol 1,4,5‐trisphosphate (InsP3) and inositol 1,3,4,5‐tetrakisphosphate (InsP4) accumulation in colonic smooth muscle. 2 In the present study, we demonstrate the existence of InsP3 and InsP4 binding sites in colonic circular smooth muscle by use of radioligand binding methods. Both [3H]‐InsP3 and [3H]‐InsP4 bound rapidly and reversibly to a single class of saturable sites in detergent‐solubilized colonic membranes with affinities of 5.04 ± 1.03 nm and 3.41 ± 0.78 nm, respectively. The density of [3H]‐InsP3 binding sites was 335.3 ± 19.3 fmol mg−1 protein which was approximately 2.5 fold greater than that of [3H]‐InsP4 sites (127.3 ± 9.1 fmol mg−1 protein). 3 The two high affinity inositol phosphate binding sites exhibited markedly different pH optima for binding of each radioligand. At pH 9.0, specific [3H]‐InsP3 binding was maximal, whereas [3H]‐InsP4 binding was only 10% that of [3H]‐InsP3. Conversely, at pH 5.0, [3H]‐InsP4 binding was maximal, while [3H]‐InsP3 binding was reduced to 15% of its binding at pH 9.0. 4 InsP3 was about 20 fold less potent (K1 = 50.7 ± 8.3 nm) than InsP4 in competing for [3H]‐InsP4 binding sites and could compete for only 60% of [3H]‐InsP4 specific binding. InsP4 was also capable of high affinity competition with [3H]‐InsP3 binding (K1 = 103.5 ± 1.5 nm), and could compete for 100% of [3H]‐InsP3 specific binding. 5 [3H]‐InsP3 binding in subcellular fractions separated by discontinuous sucrose density gradients followed NADPH‐cytochrome c reductase activity, suggesting an intracellular localization for the majority of InsP3 receptors in this tissue, whereas [3H]‐InsP4 binding appeared to be equally distributed between plasma membrane and intracellular membrane populations. 6 These results suggest the existence of distinct and specific InsP3 and InsP4 binding sites which may represent the physiological receptors for these second messengers; differences in the subcellular distribution of these receptors may contribute to differences in their putative physiological roles.

Inositolpolyphosphate binding sites and their likely role in calcium regulation in smooth muscle.

Inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) binding sites have been identified in smooth muscle and other tissues. Subcellular localization of these receptors in smooth muscle indicates that they are present in both the sarcoplasmic reticulum membrane and the plasma membrane, although the InsP3 receptor appears predominantly localized in the sarcoplasmic reticulum membrane. The heterogeneity of InsP3 binding sites is confirmed by radioligand binding and molecular cloning studies. It is now clear that InsP3, in addition to releasing intracellular Ca2+, can also stimulate Ca2+ entry across the plasma membrane. Although the mechanism of Ca2+ entry remains a matter for much debate, what is not in doubt is that increases in InsP3, perhaps acting together with InsP4, can maintain a constant influx of Ca2+ across the cell membrane. Compared to the InsP3 receptor, our understanding of the InsP4 binding site is limited. In most cases, including release of intracellular Ca2+ or Ca2+ entry, the major role of InsP4 appears to be the potentiation of the InsP3-induced response. Future studies of the InsP4 binding site by purification and molecular cloning, as well as subcellular localization, are needed to clarify the role for InsP4 in the regulation of intracellular Ca2+.