The hypothesis tested in this study is that diabetes has a different impact on an artery in which endothelium-dependent responses derive from both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) compared with responses in which NO predominates and EDHF is absent. The streptozotocin-treated rat model of diabetes was used, and the arteries were mounted on a wire myograph. In mesenteric arteries depolarized and constricted with phenylephrine, acetylcholine evoked hyperpolarization (31 +/- 2 mV) and complete relaxation; these responses were attributed to EDHF and NO. In femoral arteries, acetylcholine evoked a small, NO-mediated hyperpolarization (5 +/- 1 mV) and incomplete relaxation. Bradykinin evoked NO-dependent responses in mesenteric arteries. Whereas diabetes significantly impaired the EDHF-dependent hyperpolarization and relaxation in mesenteric arteries, NO-dependent responses in femoral and mesenteric arteries were preserved. 1-Ethyl-2-benzimidazolinone evoked hyperpolarization and relaxation in mesenteric arteries, and this was impaired in diabetes. In conclusion, NO-dependent responses are preserved in diabetes, whereas endothelial responses-dependent upon EDHF appear to be impaired. The putative channels responsible for mediating the EDHF response may be altered in diabetes.

2+ transient discharge suggesting that they may well be acting as 'point sources' of excitation to the TSMC layer. We speculate that ASMCs act as the primary pacemaker in the renal pelvis while ICC-LCs play a supportive role, but can take over pacemaking in the absence of the proximal pacemaker drive.

1. Membrane potential was recorded with intracellular microelectrodes from the smooth muscle of coronary arteries of guinea-pigs, and the responses to endothelium-derived relaxants were studied under a variety of conditions. 2. Stimulation of the endothelium with brief applications of acetylcholine or substance P evoked concentration-dependent hyperpolarizations that were complex in nature. A transient component, which is likely to result from endothelium-derived hyperpolarizing factor (EDHF), was followed by a slow component that resulted from the production of nitric oxide (NO) and a prostaglandin. 3. The ability of exogenous and endogenous NO and prostacyclin to hyperpolarize the membrane depended upon the smooth muscle being under stretch. Unstretched preparations responded to acetylcholine with only the transient component of hyperpolarization; NO and prostacyclin were without effect. 4. In stretched preparations exogenous NO and prostacyclin, and its synthetic analogue methyl prostacyclin (Iloprost), evoked hyperpolarization, and the slow component of the response induced by acetylcholine appeared. The amplitudes of these responses reached maximum when the tissues were stretched to the equivalent of approximately 50 mmHg. 5. From a resting membrane potential of -61 +/- 0.6 mV, exogenous NO and Iloprost hyperpolarized the smooth muscle to around -80 mV. The EC50 values for NO- and Iloprost-induced hyperpolarization were 2.6 x 10(-6) and 1.3 x 10(-8) M, respectively. 6. Coronary arterial smooth muscles from rats, rabbits and sheep also hyperpolarized in response to exogenous NO, although their sensitivities were less than those of preparations obtained from guinea-pigs. Iloprost hyperpolarized tissues from rabbits and sheep but not those obtained from rats. 7. It is concluded that the endothelial lining of coronary arteries can release three factors, EDHF, NO and prostacyclin, all of which can hyperpolarize the membrane of the smooth muscle. The relative proportions and significance of each factor depends on the amount of stretch, on the artery and on the species of animal.

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Pyeloureteric peristalsis has long been considered to be triggered by pacemaker atypical smooth muscle cells (SMC) located in the proximal regions of the renal pelvis. However, interstitial cells with many of the morphological features and c-Kit immuno-reactivity of interstitial cells of Cajal (ICC), the established pacemaker cells in the intestine, have been demonstrated to be present in small numbers within the ureteropelvic junction (UPJ) of many mammals. Freshly isolated ICC-like cells (ICC-LC) of the mouse UPJ also display autorhyhmicity. This review discusses the notion that ureteric peristalsis depends on the presence of both atypical SMC and ICC-LC which form separate but interconnected networks that drive electrically quiescent typical SMC. In contrast to the intestine or prostate, all regenerative potential discharge in the mouse UPJ is abolished by the L-type Ca 2+ channel blocker nifedipine revealing a fundamental pacemaker signal. Whether these pacemaker transients arise from atypical SMC or ICC-LC or both has yet to be established. We speculate that the presence of spontaneously active ICC-LC in the distal regions of the UPJ maintains rudimentary peristaltic waves and movement of urine towards the bladder after pyeloureteral obstruction or pyeloplasty and disconnection from the proximal pacemaker drive.

1. Membrane potential and tension were measured simultaneously in ring segments of main coronary artery of guinea-pigs. The synthetic thromboxane A2 analogue U46619 depolarized the tissues from -58 +/- 2 to -40 +/- 1 mV and increased tension by 12 +/- 1 mN mm-1. Nitric oxide (NO) and Iloprost, the stable analogue of prostacyclin, evoked hyperpolarization and relaxation. 2. The concentration of NO required to evoke half-maximal hyperpolarization (EC50 of 2 x 10(-5) M) was 40-fold higher than that which was required to induce relaxation (EC50 of 5 x 10(-7) M). The EC50 for Iloprost-induced hyperpolarization (3 x 10(-8) M) was similar to that for relaxation (4 x 10(-8) M). 3. Glibenclamide (10(-6) M) abolished the hyperpolarization in response to both NO and Iloprost but was without effect on the amplitudes of the relaxations over the complete concentration-response curves. 4. Acetylcholine evoked concentration-dependent hyperpolarization and relaxation in the presence of N omega-nitro-L-arginine methyl ester (NAME; 10(-5) M) and indomethacin (10(-6) M), and these responses were attributed to endothelium-derived hyperpolarizing factor (EDHF). The hyperpolarization produced by EDHF always preceded relaxation, and relaxation never occurred at concentrations of acetylcholine that were insufficient to evoke hyperpolarization. 5. The concentration-hyperpolarization and concentration-relaxation curves in response to acetylcholine were not affected by glibenclamide or barium (1-3 mM) but were shifted to the right 4- and 5-fold, respectively, by 1 mM tetraethylammonium. The hyperpolarization and relaxation evoked by acetylcholine were also reduced in a parallel manner when the potassium concentration in the superfusate was increased. 6. Hyperpolarizing current steps, applied to spiral strips of coronary artery denuded of endothelium and depolarized and constricted with U46619, caused relaxation. The relationship between hyperpolarization and relaxation evoked electronically was similar to that which was due to EDHF in intact tissues stimulated with acetylcholine. 7. It is concluded that the ability of NO or Iloprost to relax guinea-pig coronary artery does not depend upon hyperpolarization of the smooth muscle. In contrast, hyperpolarization is likely to play a major, if not the only, role in the relaxation in response to EDHF in this tissue.

The recent introduction of technologies capable of reprogramming human somatic cells into induced pluripotent stem (iPS) cells offers a unique opportunity to study many aspects of neurodegenerative diseases in vitro that could ultimately lead to novel drug development and testing. Here, we report for the first time that human dermal fibroblasts from a patient with relapsing-remitting Multiple Sclerosis (MS) were reprogrammed to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4, and c-MYC). The MSiPS cell lines resembled human embryonic stem (hES) cell-like colonies in morphology and gene expression and exhibited silencing of the retroviral transgenes after four passages. MSiPS cells formed embryoid bodies that expressed markers of all three germ layers by immunostaining and Reverse Transcriptase (RT)-PCR. The injection of undifferentiated iPS cell colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. The MSiPS cells were successfully differentiated into mature astrocytes, oligodendrocytes and neurons with normal karyotypes. Although MSiPS-derived neurons displayed some differences in their electrophysiological characteristics as compared to the control cell line, they exhibit properties of functional neurons, with robust resting membrane potentials, large fast tetrodotoxin-sensitive action potentials and voltage-gated sodium currents. This study provides for the first time proof of concept that disease cell lines derived from skin cells obtained from an MS patient can be generated and successfully differentiated into mature neural lineages. This represents an important step in a novel approach for the study of MS pathophysiology and potential drug discovery.

Background and purpose: Electrically active atypical smooth muscle cells (ASMCs) within the renal pelvis have long been considered to act as pacemaker cells driving pelviureteric peristalsis. We have investigated the role of Ca2+ entry and uptake into and release from internal stores in the generation of Ca2+ transients and spontaneous transient depolarizations (STDs) in ASMCs. Experimental approach: The electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and interstitial cells of Cajal‐like cells (ICC‐LCs) were recorded using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo‐4. Results: In 1 μM nifedipine, high frequency (10–30 min−1) Ca2+ transients and STDs were recorded in ASMCs, while ICC‐LCs displayed low frequency (1–3 min−1) Ca2+ transients. All spontaneous electrical activity and Ca2+ transients were blocked upon removal of Ca2+ from the bathing solution, blockade of Ca2+ store uptake with cyclopiazonic acid (CPA) and with 2‐aminoethoxy‐diphenylborate (2‐APB). STD amplitudes were reduced upon removal of the extracellular Na+ or blockade of IP3 dependent Ca2+ store release with neomycin or U73122. Blockade of ryanodine‐sensitive Ca2+ release blocked ICC‐LC Ca2+ transients but only reduced Ca2+ transient discharge in ASMCs. STDs in ASMCS were also little affected by DIDS, La3+, Gd3+ or by the replacement of extracellular Cl‐ with isethionate. Conclusions: ASMCs generated Ca2+ transients and cation‐selective STDs via mechanisms involving Ca2+ release from IP3‐dependent Ca2+ stores, STD stimulation of TSMCs was supported by Ca2+ entry through L type Ca2+ channels and Ca2+ release from ryanodine‐sensitive stores. British Journal of Pharmacology (2007) 152, 1248–1259; doi:; published online 29 October 2007