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1.
Pflugers Arch ; 473(12): 1925-1938, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34596752

RESUMO

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5 mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300 µm dia.) exhibited SPCs, while distal segments (350-500 µm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1 µM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1 µM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10 µM). SPCs were also inhibited by caffeine (1 µM), 2-APB (100 µM), niflumic acid (100 µM), or by lowering extracellular [Cl-] from 134.4 to 12.4 mM but not by ryanodine (25 µM) or tetracaine (100 µM). Electrical field stimulation (EFS) at 2 Hz for 60 s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.


Assuntos
Epididimo/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Epididimo/efeitos dos fármacos , Epididimo/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Nifedipino/farmacologia , Norepinefrina/farmacologia , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Rianodina/farmacologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo
2.
3.
Adv Exp Med Biol ; 1124: 77-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183823

RESUMO

The peristaltic pressure waves in the renal pelvis that propel urine expressed by the kidney into the ureter towards the bladder have long been considered to be 'myogenic', being little affected by blockers of nerve conduction or autonomic neurotransmission, but sustained by the intrinsic release of prostaglandins and sensory neurotransmitters. In uni-papilla mammals, the funnel-shaped renal pelvis consists of a lumen-forming urothelium and a stromal layer enveloped by a plexus of 'typical' smooth muscle cells (TSMCs), in multi-papillae kidneys a number of minor and major calyces fuse into a large renal pelvis. Electron microscopic, electrophysiological and Ca2+ imaging studies have established that the pacemaker cells driving pyeloureteric peristalsis are likely to be morphologically distinct 'atypical' smooth muscle cells (ASMCs) that fire Ca2+ transients and spontaneous transient depolarizations (STDs) which trigger propagating nifedipine-sensitive action potentials and Ca2+ waves in the TSMC layer. In uni-calyceal kidneys, ASMCs predominately locate on the serosal surface of the proximal renal pelvis while in multi-papillae kidneys they locate within the sub-urothelial space. 'Fibroblast-like' interstitial cells (ICs) located in the sub-urothelial space or adventitia are a mixed population of cells, having regional and species-dependent expression of various Cl-, K+, Ca2+ and cationic channels. ICs display asynchronous Ca2+ transients that periodically synchronize into bursts that accelerate ASMC Ca2+ transient firing. This review presents current knowledge of the architecture of the proximal renal pelvis, the role Ca2+ plays in renal pelvis peristalsis and the mechanisms by which ICs may sustain/accelerate ASMC pacemaking.


Assuntos
Cálcio/fisiologia , Células Intersticiais de Cajal/fisiologia , Canais Iônicos/fisiologia , Pelve Renal/fisiologia , Contração Muscular , Peristaltismo , Animais , Ureter
4.
Adv Exp Med Biol ; 1124: 103-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183824

RESUMO

The ureter acts as a functional syncytium and is controlled by a propagating plateau-type action potential (AP) which gives rise to a wave of contraction (ureteral peristalsis) via a process called excitation-contraction (E-C)coupling. The second messenger Ca2+ activates Ca2+/calmodulin-dependent myosin light chain kinase-dependent phosphorylation of 20-kDa regulatory light chains of myosin which leads to ureteric contraction. Ca2+ entry from the extracellular space via voltage-gated L-type Ca2+ channels (VGCCs) provides the major source of activator Ca2+, responsible for generation of both the AP and a Ca2+ transient that appears as an intercellular Ca2+ wave. The AP, inward Ca2+ current, Ca2+ transient and twitch contraction are all fully blocked by the selective L-type Ca2+ channel blocker nifedipine. Ca2+ entry via VGCCs, coupled to activation of Ca2+-sensitive K+ (KCa) or Cl- (ClCa) channels, acts as a negative or positive feedback mechanism, respectively, to control excitability and the amplitude and duration of the plateau component of the AP, Ca2+ transient and twitch contraction. The ureter, isolated from the pelvis, is not spontaneously active. However, spontaneous activity can be initiated in the proximal and distal ureter by a variety of biological effectors such as neurotransmitters, paracrine, endocrine and inflammatory factors. Applied agonists depolarise ureteric smooth muscles cells to threshold of AP activation, initiating propagating intercellular AP-mediated Ca2+ waves to produce antegrade and/or retrograde ureteric peristalsis. Several mechanisms have been proposed to describe agonist-induced depolarization of ureteric smooth muscle, which include suppression of K+ channels, stimulation of ClCa current and activation of non-selective cation receptor/store operated channels.


Assuntos
Sinalização do Cálcio , Canais Iônicos/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Peristaltismo , Ureter/fisiologia , Cálcio/fisiologia , Humanos
5.
J Smooth Muscle Res ; 53(0): 57-72, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28652517

RESUMO

The prostate is a gland whose secretions contribute to the seminal fluids ejaculated upon activation of autonomic sympathetic nerves. In elder males, the prostate undergoes an increase in stroma mass and myogenic tone, leading to benign prostatic hyperplasia that occludes the proximal urethra and the presentation of various lower urinary tract symptoms that decrease their quality of life. This review summarises the role of prostatic interstitial cells (PICs) in the generation of the spontaneous tone in the prostate. It presents current knowledge of the role of Ca2+ plays in PIC pacemaking, as well as the mechanisms by which this spontaneous activity triggers slow wave generation and stromal contraction. PICs display a small T-type Ca2+ current (ICaT) and a large L-type Ca2+ current (ICaL). In contrast to other interstitial cells in the urinary and gastrointestinal tracts, spontaneous Ca2+ signalling in PICs is uniquely dependent on Ca2+ influx through ICaL channels. A model of prostatic pacemaking is presented describing how ICaL can be triggered by an initial membrane depolarization evoked upon the selective opening of Ca2+-activated Cl- channels by Ca2+ flowing only through ICaT channels. The resulting current flow through ICaL results in release of Ca2+ from internal stores and the summation of Cl--selective spontaneous transient depolarizations (STDs) to form pacemaker potentials that propagate passively into the prostatic stroma to evoke regenerative action potentials and excitation-contraction coupling.


Assuntos
Próstata/citologia , Próstata/fisiologia , Potenciais de Ação/fisiologia , Animais , Relógios Biológicos/fisiologia , Cálcio/metabolismo , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Canais de Cloreto/fisiologia , Fenômenos Eletrofisiológicos , Gerbillinae , Cobaias , Humanos , Técnicas In Vitro , Canais Iônicos/fisiologia , Masculino , Camundongos , Hiperplasia Prostática/etiologia , Ratos , Sistema Nervoso Simpático/fisiologia , Transmissão Sináptica/fisiologia
6.
Pflugers Arch ; 469(5-6): 797-813, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28054154

RESUMO

Typical and atypical smooth muscle cells (TSMCs and ASMCs, respectively) and interstitial cells (ICs) within the pacemaker region of the mouse renal pelvis were examined using focused ion beam scanning electron (FIB SEM) tomography, immunohistochemistry and Ca2+ imaging. Individual cells within 500-900 electron micrograph stacks were volume rendered and associations with their neighbours established. 'Ribbon-shaped', Ano1 Cl- channel immuno-reactive ICs were present in the adventitia and the sub-urothelial space adjacent to the TSMC layer. ICs in the proximal renal pelvis were immuno-reactive to antibodies for CaV3.1 and hyperpolarization-activated cation nucleotide-gated isoform 3 (HCN3) channel sub-units, while basal-epithelial cells (BECs) were intensely immuno-reactive to Kv7.5 channel antibodies. Adventitial to the TSMC layer, ASMCs formed close appositions with TSMCs and ICs. The T-type Ca2+channel blocker, Ni2+ (10-200 µM), reduced the frequency while the L-type Ca2+ channel blocker (1 µM nifedipine) reduced the amplitude of propagating Ca2+ waves and contractions in the TSMC layer. Upon complete suppression of Ca2+ entry through TSMC Ca2+ channels, ASMCs displayed high-frequency (6 min-1) Ca2+ transients, and ICs distributed into two populations of cells firing at 1 and 3 min-1, respectively. IC Ca2+ transients periodically (every 3-5 min-1) summed into bursts which doubled the frequency of ASMC Ca2+ transient firing. Synchronized IC bursting and the acceleration of ASMC firing were inhibited upon blockade of HCN channels with ZD7288 or cell-to-cell coupling with carbenoxolone. While ASMCs appear to be the primary pacemaker driving pyeloureteric peristalsis, it was concluded that sub-urothelial HCN3(+), CaV3.1(+) ICs can accelerate ASMC Ca2+ signalling.


Assuntos
Sinalização do Cálcio , Células Intersticiais de Cajal/metabolismo , Miócitos de Músculo Liso/metabolismo , Peristaltismo , Fenômenos Fisiológicos do Sistema Urinário , Animais , Canais de Cálcio/metabolismo , Tomografia com Microscopia Eletrônica , Feminino , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Células Intersticiais de Cajal/fisiologia , Células Intersticiais de Cajal/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/ultraestrutura , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sistema Urinário/metabolismo , Sistema Urinário/ultraestrutura
7.
Clin Exp Pharmacol Physiol ; 43(5): 535-42, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26876143

RESUMO

The action of angiotensin II (AngII) on the Ca(2+) signals driving pyeloureteric peristalsis was investigated using both conventional and angiotensin receptor (ATr) ATr1A and ATr2 knockout ((-/-)) mice. Contractility in the renal pelvis of adult ATr1A(-/-) and ATr2(-/-) mice was compared to their respective wildtype (ATr1A(+/+) and ATr2(+/+)) controls of the same genetic background (FVB/N and C57Bl/6 respectively) using video microscopy. The effects of AngII on the Ca(2+) signals in typical and atypical smooth muscle cells (TSMCs and ASMCs, respectively) within the pelvic wall of conventional mice were recorded using Fluo-4 Ca(2+) imaging. Compared to ATr1A(+/+) , ATr2(+/+) and ATr2(-/-) mice, kidneys of the ATr1A(-/-) mouse were mildly-to-severely hydronephrotic, associated with an enlarged calyx, an atrophic papilla and a hypoplastic renal pelvis. Contraction frequencies in the renal pelvis of moderately hydronephrotic ATr1A(-/-) and unaffected ATr2(-/-) mice were not significantly different from their ATr1A(+/+), ATr2(+/+) controls. No contractions were observed in severely-hydronephrotic ATr1A(-/-) kidneys. AngII increased the spontaneous contraction frequency of the renal pelvis in ATr1A(+/+), ATr2(+/+) and ATr2(-/-) mice, but had little effect on the contractions in the mildly-hydronephrotic ATr1A(-/-) renal pelvis. The ATr1 blocker, candesartan prevented the positive chronotropic effects of AngII. AngII increased the frequency and synchronicity of Ca(2+) transients in both TSMCs and ASMCs. It was concluded that the hydronephrosis observed in ATr1A(-/-) mouse kidneys does not arise from a failure in the development of the essential pacemaker and contractile machinery driving pyeloureteric peristalsis.


Assuntos
Técnicas de Inativação de Genes , Hidronefrose/genética , Hidronefrose/fisiopatologia , Pelve Renal/fisiopatologia , Peristaltismo/genética , Receptor Tipo 1 de Angiotensina/deficiência , Receptor Tipo 1 de Angiotensina/genética , Angiotensina II/farmacologia , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Feminino , Hidronefrose/patologia , Pelve Renal/efeitos dos fármacos , Pelve Renal/patologia , Masculino , Camundongos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Peristaltismo/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/deficiência , Receptor Tipo 2 de Angiotensina/genética
8.
J Physiol ; 594(3): 555-65, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26607499

RESUMO

The microvasculature plays a primary role in the interchange of substances between tissues and the circulation. In visceral organs that undergo considerable distension upon filling, the microvasculature appears to display intrinsic contractile properties to maintain their flow. Submucosal venules in the bladder or gastrointestinal tract generate rhythmic spontaneous phasic constrictions and associated Ca(2+) transients. These events are initiated within either venular pericytes or smooth muscle cells (SMCs) arising from spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) and the opening of Ca(2+) -activated chloride channels (CaCCs) that trigger Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (VDCCs). L-type VDCCs also play a critical role in maintaining synchrony within the contractile mural cells. In the stomach myenteric layer, spontaneous Ca(2+) transients originating in capillary pericytes appear to spread to their neighbouring arteriolar SMCs. Capillary Ca(2+) transients primarily rely on SR Ca(2+) release, but also require Ca(2+) influx through T-type VDCCs for their synchrony. The opening of T-type VDCCs also contribute to the propagation of Ca(2+) transients into SMCs. In visceral microvasculature, pericytes act as either spontaneously active contractile machinery of the venules or as pacemaker cells generating synchronous Ca(2+) transients that drive spontaneous contractions in upstream arterioles. Thus pericytes play different roles in different vascular beds in a manner that may well depend on the selective expression of T-type and L-type Ca(2+) channels.


Assuntos
Canais de Cálcio/fisiologia , Microvasos/fisiologia , Pericitos/fisiologia , Animais , Trato Gastrointestinal/irrigação sanguínea , Trato Gastrointestinal/fisiologia , Humanos , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/fisiologia
9.
BJU Int ; 114(3): 436-46, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24467706

RESUMO

OBJECTIVE: To investigate the expression of 'T-type' and 'L-type' voltage-operated Ca(2) (+) channels in single interstitial cells of the guinea-pig prostate. MATERIAL AND METHODS: Whole-cell and perforated patch-clamp techniques were applied to prostatic interstitial cells (PICs) dispersed using collagenase. RESULTS: In contrast to prostatic myocytes, PICs under voltage clamp and filled with K(+) (130 mm) were distinguished by the absence of a voltage-operated transient outward K(+) current or spike discharge upon membrane depolarisation when under current clamp. Depolarisation of Cs(+) -filled PICs evoked an inward current at potentials positive to -60 mV, which peaked in amplitude near 0 mV. This inward current increased when Ba(2+) (5 mm) replaced the external Ca(2) (+) (1.5 mm) and displayed a variable sensitivity to the inhibitory actions of conditioning depolarisations to -40 mV applied before the test depolarisation or to 1 µm nifedipine, the 'L-type' Ca(2) (+) channel blocker. A residual inward current recorded in nifedipine was blocked by 10 µm Ni(2) (+) . Cs(+) -filled PICs also displayed a slowly inactivating outward current that was little affected by nifedipine, reduced by the Cl(-) channel blocker, niflumic acid (10 µm) and blocked by Ba(2) (+) or a conditioning depolarisation. CONCLUSION: PICs express both a small 'T-type' Ca(2) (+) channel current (ICa ) and a large 'L-type' ICa . Ca(2) (+) influx through 'T-type' ICa was an essential trigger for the activation of a Ca(2) (+) -activated Cl(-) -selective current. The dependence of PIC Ca(2) (+) signalling on 'T-type' and 'L-type' ICa is unique compared with other interstitial cells of the urogenital tract and may well be pharmaceutically exploitable.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Canais de Cloreto/metabolismo , Células Intersticiais de Cajal/fisiologia , Miócitos de Músculo Liso/fisiologia , Nifedipino/farmacologia , Próstata/metabolismo , Animais , Canais de Cloreto/efeitos dos fármacos , Potenciais Evocados/efeitos dos fármacos , Cobaias , Masculino , Técnicas de Patch-Clamp , Próstata/citologia
10.
BJU Int ; 112(4): E398-405, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23879919

RESUMO

UNLABELLED: What's known on the subject? and what does the study add?: Several studies have examined the functional role of tyrosine kinase receptors in the generation of spontaneous activity in various segments of the gastrointestinal and urogenital tracts through the application of its inhibitor, imatinib mesylate (Glivec®), but results are fairly inconsistent. This is the first study detailing the effects of imatinib mesylate on the spontaneous activity in the young and ageing prostate gland. As spontaneous electrical activity underlies the spontaneous rhythmic prostatic contractions that occur at rest, elucidating the mechanisms involved in the regulation of the spontaneous electrical activity and the resultant phasic contractions could conceivably lead to the identification of better targets and the development of more specific therapeutic agents to treat prostate conditions. OBJECTIVE: To investigate the effect of imatinib mesylate, a tyrosine kinase receptor inhibitor, in the generation of spontaneous electrical and contractile activity in the young and ageing guinea-pig prostate. MATERIALS AND METHODS: Standard tension and intracellular recording were used to measure spontaneous contractions and slow waves, respectively from the guinea-pig prostate at varying concentrations of imatinib mesylate (1-50 µm). RESULTS: Imatinib mesylate (1-10 µm), did not significantly affect slow waves recorded in the prostate of both age groups but at 50 µm, the amplitude of slow waves from the ageing guinea-pig prostate was significantly reduced (P < 0.05, n = 5). In contrast, the amplitude of contractions across all concentrations in the young guinea-pig prostate was reduced to between 35% and 41% of control, while the frequency was reduced to 15.7% at 1 µm (n = 7), 49.8% at 5 µm (n = 10), 46.2% at 10 µm (n = 7) and 53.1% at 50 µm (n = 5). Similarly, imatinib mesylate attenuated the amplitude and slowed the frequency of contractions in ageing guinea-pigs to 5.15% and 3.3% at 1 µm (n = 6); 21.1% and 20.8% at 5 µm (n = 8); 58.4% and 8.8% at 10 µm (n = 11); 72.7% and 60% at 50 µm (n = 5). CONCLUSIONS: A significant reduction in contractions but persistence of slow waves suggests imatinib mesylate may affect the smooth muscle contractile mechanism. Imatinib mesylate also significantly reduced contractions in the prostates of younger guinea pigs more than older ones, which is consistent with the notion that the younger guinea-pig prostate is more reliant on the tyrosine-dependent pacemaker ability of interstitial cells of Cajal-like prostatic interstitial cells.


Assuntos
Benzamidas/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Piperazinas/farmacologia , Próstata/efeitos dos fármacos , Próstata/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Cobaias , Mesilato de Imatinib , Masculino
11.
J Pathol ; 227(4): 499-510, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22488641

RESUMO

Crim1 hypomorphic (Crim1(KST264/KST264)) mice display progressive renal disease characterized by glomerular defects, leaky peritubular vasculature, and progressive interstitial fibrosis. Here we show that 27% of these mice also present with hydronephrosis, suggesting obstructive nephropathy. Dynamic magnetic resonance imaging using Magnevist showed fast development of hypo-intense signal in the kidneys of Crim1(KST264/KST264) mice, suggesting pooling of filtrate within the renal parenchyma. Rhodamine dextran (10 kDa) clearance was also delayed in Crim1(KST264/KST264) mice. Pyeloureteric peristalsis, while present, was less co-ordinated in Crim1(KST264/KST264) mice. However, isolated renal pelvis preparations suggest normal pelvic smooth muscle contractile responses. An analysis of maturation during the immediate postnatal period [postnatal day (P) 0-15] revealed defects in papillary extension in Crim1({KST264/KST264) mice. While Crim1 expression is weak in pelvic smooth muscle, strong expression is seen in the interstitium and loops of Henle of the extending papilla, commencing at the tip of the P1 papilla and disseminating throughout the papilla by P15. These results, as well as implicating Crim1 in papillary extension and pelvic smooth muscle contractility, highlight the previously unrecognized association between defects in papillary development and progression to chronic kidney disease later in life.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/genética , Hidronefrose/congênito , Nefropatias/congênito , Medula Renal/anormalidades , Medula Renal/fisiopatologia , Mutação/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Dextranos/metabolismo , Hidronefrose/metabolismo , Hidronefrose/fisiopatologia , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Medula Renal/metabolismo , Alça do Néfron/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Modelos Animais , Contração Muscular/fisiologia , Miócitos de Músculo Liso/patologia , Rodaminas/metabolismo
12.
J Urol ; 187(6): 2254-60, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22503041

RESUMO

PURPOSE: We investigated nitric oxide mediated inhibition of spontaneous activity recorded in young and aging guinea pig prostates. MATERIALS AND METHODS: Conventional intracellular microelectrode and tension recording techniques were used. RESULTS: The nitric oxide donor sodium nitroprusside (10 µM) abolished spontaneous contractions and slow wave activity in 5 young and 5 aging prostates. Upon adding the nitric oxide synthase inhibitor L-NAME (10 µM) the frequency of spontaneous contractile and electrical activity was significantly increased in each age group. This increase was significantly larger in 4 to 8 preparations of younger vs aging prostates (about 40% to 50% vs about 10% to 20%, 2-way ANOVA p<0.01). Other measured parameters, including the duration, amplitude and membrane potential of spontaneous electrical and contractile activity, were not altered from control values. The guanylate cyclase inhibitor ODQ (10 µM) significantly increased the frequency of spontaneous activity by 10% to 30% in 6 young guinea pig prostates (Student paired t test p<0.05). However, it had no effect on aging prostates. The cGMP analogue 8-Br-GMP (1 µM) and the PDE5 inhibitor dipyridamole (1 µM) significantly decreased the frequency of contractile activity by about 70% in 4 to 9 young and older prostates (Student paired t test p<0.05). CONCLUSIONS: The decrease in the response to L-NAME in spontaneous contractile and slow wave activity in aging prostate tissue compared to that in young prostates suggests that with age there is a decrease in nitric oxide production. This may further explain the increase in prostatic smooth muscle tone observed in age related prostate specific conditions, such as benign prostatic hyperplasia.


Assuntos
Óxido Nítrico/biossíntese , Próstata/metabolismo , Envelhecimento/metabolismo , Animais , Cobaias , Masculino , Modelos Animais , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Óxido Nítrico/metabolismo , Próstata/fisiologia , Transdução de Sinais
13.
Br J Pharmacol ; 165(7): 2389-408, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22014103

RESUMO

BACKGROUND AND PURPOSE: Although atypical smooth muscle cells (SMCs) in the proximal renal pelvis are thought to generate the pacemaker signals that drive pyeloureteric peristalsis, their location and electrical properties remain obscure. EXPERIMENTAL APPROACH: Standard patch clamp, intracellular microelectrode and immunohistochemistry techniques were used. To unequivocally identify SMCs, transgenic mice with enhanced yellow fluorescent protein (eYFP) expressed in cells containing α-smooth muscle actin (α-SMA) were sometimes used. KEY RESULTS: Atypical SMCs were distinguished from typical SMCs by the absence of both a transient 4-aminopyridine-sensitive K(+) current (I(KA) ) and spontaneous transient outward currents (STOCs) upon the opening of large-conductance Ca(2+) -activated K(+) (BK) channels. Many typical SMCs displayed a slowly activating, slowly decaying Cl(-) current blocked by niflumic acid (NFA). Immunostaining for K(V) 4.3 and ANO1/ TMEM16A Cl(-) channel subunits co-localized with α-SMA immunoreactive product predominately in the distal renal pelvis. Atypical SMCs fired spontaneous inward currents that were either selective for Cl(-) and blocked by NFA, or cation-selective and blocked by La(3+) . α-SMA(-) interstitial cells (ICs) were distinguished by the presence of a Xe991-sensitive K(V) 7 current, BK channel STOCs and Cl(-) selective, NFA-sensitive spontaneous transient inward currents (STICs). Intense ANO1/ TMEM16A and K(V) 7.5 immunostaining was present in Kit(-) α-SMA(-) ICs in the suburothelial and adventitial regions of the renal pelvis. CONCLUSIONS AND IMPLICATIONS: We conclude that K(V) 4.3(+) α-SMA(+) SMCs are typical SMCs that facilitate muscle wall contraction, that ANO1/ TMEM16A and K(V) 7.5 immunoreactivity may be selective markers of Kit(-) ICs and that atypical SMCs which discharge spontaneous inward currents are the pelviureteric pacemakers.


Assuntos
Canais de Cloreto/metabolismo , Pelve Renal/citologia , Pelve Renal/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio/metabolismo , Actinas/metabolismo , Animais , Anoctamina-1 , Biomarcadores/metabolismo , Feminino , Canais de Potássio KCNQ/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Miócitos de Músculo Liso/classificação , Miócitos de Músculo Liso/citologia , Técnicas de Patch-Clamp , Canais de Potássio Shal/metabolismo
14.
J Urol ; 186(6): 2478-86, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22019167

RESUMO

PURPOSE: We investigated whether prostate interstitial cells generate spontaneous Ca(2+) oscillation, a proposed mechanism underlying pacemaker potentials to drive spontaneous activity in stromal smooth muscle cells. MATERIALS AND METHODS: Intracellular free Ca(2+) in portions of guinea pig prostate and freshly isolated, single prostate interstitial cells were visualized using fluo-4 Ca(2+) fluorescence. Spontaneous electrical activity was recorded in situ with intracellular microelectrodes. RESULTS: In whole tissue preparations spontaneous Ca(2+) flashes firing synchronously across all smooth muscle cells within the field of view resulted in muscle wall contractions. Nonpropagating Ca(2+) waves were also recorded in individual smooth muscle cells. Nifedipine (Sigma®) (1 µM) largely decreased or abolished these Ca(2+) flashes and suppressed slow wave discharge upon blockade of their superimposed action potentials. Isolated prostate interstitial cells were readily distinguished from smooth muscle cells by their spiky processes and lack of contraction during intracellular Ca(2+) increases. Prostate interstitial cells generated spontaneous Ca(2+) transients in the form of whole cell flashes, intracellular Ca(2+) waves or localized Ca(2+) sparks. All 3 Ca(2+) signals were abolished by nicardipine (1 µM), cyclopiazonic acid (10 µM), caffeine (Sigma) (10 mM) or extracellular Ca(2+) removal. CONCLUSIONS: Prostate interstitial cells generate spontaneous Ca(2+) transients that occur at a frequency comparable to Ca(2+) flashes in situ or slow waves relying on functional internal Ca(2+) stores. However, unlike other interstitial cells in the urinary tract, Ca(2+) influx through L-type Ca(2+) channels is fundamental to Ca(2+) transient firings in prostate interstitial cells. Thus, it is not possible to conclude that prostate interstitial cells are responsible for pacemaker potential generation.


Assuntos
Sinalização do Cálcio , Células Intersticiais de Cajal/fisiologia , Próstata/metabolismo , Animais , Cobaias , Masculino
15.
Stem Cells ; 29(3): 462-73, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21425409

RESUMO

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.


Assuntos
Linhagem da Célula/genética , Rastreamento de Células/métodos , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/genética , Prosencéfalo/embriologia , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/citologia , Citometria de Fluxo/métodos , Genes Reporter , Humanos , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Neurogênese/genética , Neurogênese/fisiologia , Proteínas Nucleares/metabolismo , Prosencéfalo/citologia , Prosencéfalo/fisiologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo
17.
J Urol ; 185(1): 347-51, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21075393

RESUMO

PURPOSE: We focused on the current opinion on mechanisms generating stromal tone in the prostate gland. MATERIALS AND METHODS: We selected the guinea pig as the main species for investigation since its prostate has a high proportion of smooth muscle that undergoes age related changes similar in many respects to that in humans. The main techniques that we used were tension recording and electrophysiology. RESULTS: We previously reported distinct electrical activity and cell types in the prostate, and speculated on their functional roles. We believe that a specialized group of c-kit immunoreactive prostatic interstitial cells that lie between glandular epithelium and smooth muscle stroma have a role similar to that of gastrointestinal interstitial cells of Cajal, generating the pacemaker signal that manifests as slow wave activity and triggers contraction in smooth muscle cells in guinea pig prostates. CONCLUSIONS: Since changes in muscle tone are involved in the etiology of age dependent prostate specific conditions such as benign prostatic hyperplasia, knowledge of the electrical properties of the various prostatic cell types and their interactions with each other, with nerves and with the hormonal environment, and how these factors change with age is of considerable medical importance.


Assuntos
Próstata/citologia , Próstata/fisiologia , Animais , Relógios Biológicos , Cobaias , Masculino , Contração Muscular/fisiologia , Músculo Liso/citologia , Músculo Liso/fisiologia , Próstata/inervação
18.
Br J Pharmacol ; 161(3): 680-94, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20880405

RESUMO

BACKGROUND AND PURPOSE: Although spontaneous Ca(2+) waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca(2+) release, the interactions among mitochondrial Ca(2+) buffering, cellular energetics and ER Ca(2+) release in determining the spatiotemporal dynamics of intracellular Ca(2+) remain to be elucidated. EXPERIMENTAL APPROACH: Spontaneous Ca(2+) transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca(2+) imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red. KEY RESULTS: Spontaneous Ca(2+) waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca(2+) dynamics were characterized by a gradual rise in basal Ca(2+) that preceded each regenerative Ca(2+) transient. Caffeine evoked oscillatory Ca(2+) waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca(2+) wave generation with a rise in basal Ca(2+), and subsequent caffeine evoked a single rudimentary Ca(2+) transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca(2+) and abolished Ca(2+) waves. However, caffeine still induced oscillatory Ca(2+) transients. Mitochondrial Ca(2+) uptake inhibition with RU360 attenuated Ca(2+) wave amplitudes, while mitochondrial Ca(2+) efflux inhibition with CGP37157 suppressed the initial Ca(2+) rise to reduce Ca(2+) wave frequency. CONCLUSIONS AND IMPLICATIONS: Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca(2+) wave generation and may regulate ER Ca(2+) release frequency by buffering Ca(2+) within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca(2+) buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.


Assuntos
Sinalização do Cálcio/fisiologia , Células Intersticiais de Cajal/fisiologia , Mitocôndrias/fisiologia , Uretra/fisiologia , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Desoxiglucose/farmacologia , Retículo Endoplasmático/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Hidrazonas/farmacologia , Indóis/farmacologia , Células Intersticiais de Cajal/metabolismo , Masculino , Coelhos , Compostos de Rutênio/farmacologia , Rianodina/farmacologia , Tiazepinas/farmacologia , Uretra/citologia , Uretra/efeitos dos fármacos , Uretra/metabolismo
19.
Br J Pharmacol ; 161(8): 1692-707, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20735413

RESUMO

BACKGROUND AND PURPOSE: To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs. EXPERIMENTAL APPROACH: Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18ß glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2-5 months) and old (9-16 months) guinea pigs. KEY RESULTS: 18ß Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues. CONCLUSION AND IMPLICATIONS: When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.


Assuntos
Conexina 43/fisiologia , Contração Muscular/fisiologia , Próstata/fisiologia , Fatores Etários , Animais , Carbenoxolona/farmacologia , Conexina 43/metabolismo , Junções Comunicantes/efeitos dos fármacos , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Cobaias , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Octanóis/farmacologia , Próstata/efeitos dos fármacos
20.
Kidney Int ; 77(6): 483-5, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20186160

RESUMO

Although it has been recognized for more than 140 years that the pacemaker driving pyeloureteric peristalsis resides within the kidney, the cellular mechanisms underlying this autorhythmicity have been little investigated. The demonstration by Hurtado et al. of a role of hyperpolarization-activated cation channels in the maintenance of coordinated propagating contractions in the mouse renal pelvis raises a number of intriguing possibilities in addition to the provided correlation with 'pacemaker' function as in the heart and neurons.


Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Pelve Renal/fisiologia , Peristaltismo/fisiologia , Canais de Potássio/fisiologia , Animais , Cátions , Coração/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Camundongos , Camundongos Knockout , Neurônios/fisiologia
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