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Biomedical subjects

A J Kanai

Publications and source records attributed to A J Kanai.

13 recordsLinked to original sources
PubMed2005

Altered inducible nitric oxide synthase expression and nitric oxide production in the bladder of cats with feline interstitial cystitis.

PURPOSE: Alterations in nitric oxide (NO) levels have been demonstrated in some humans with interstitial cystitis (IC) as well as in chemically induced animal models of cystitis. Thus, in the current study we investigated whether inducible NO synthase (iNOS) mediated NO production is altered in the bladder of cats with a naturally occurring model of IC termed feline IC (FIC). MATERIALS AND METHODS: We examined iNOS expression using Western immunoblotting and baseline NO production using an NO microsensor from smooth muscle and mucosal bladder strips in 9 healthy cats and 6 diagnosed with FIC. RESULTS: There was a significant increase in baseline NO production in cats with FIC compared with that in healthy cats in smooth muscle and mucosal strips. This production was not ablated in the absence of extracellular Ca (100 microM egtazic acid) or following incubation with the calmodulin antagonist trifluoroperazine (20 microM), indicating iNOS mediated Ca independent NO production. Release was significantly decreased following incubation with the NOS antagonist L-NAME (N-nitro-L-arginine methyl ester) (100 microM). Furthermore, immunoblotting revealed a trend toward increased iNOS expression in smooth muscle and mucosal strips from FIC cats but not from healthy cats. CONCLUSIONS: In light of previous findings that the barrier property of the urothelial surface is disrupted in FIC and iNOS mediated increase in NO alters barrier function in other types of epithelium our findings suggest that iNOS dependent NO production may have a role in epithelial barrier dysfunction in FIC.

Animals↗
PubMed2003

Feline interstitial cystitis results in mechanical hypersensitivity and altered ATP release from bladder urothelium.

ATP can be released from a variety of cell types by mechanical stimulation; however, the mechanism for this release and the influence of pathology are not well understood. The present study examined intracellular signaling mechanisms involved in swelling-evoked (exposure to a hypotonic solution) release of ATP in urothelial cells from normal cats and cats diagnosed with interstitial cystitis (feline interstitial cystitis; FIC). Using the luciferin-luciferase bioluminescent assay, we demonstrate that swelling-evoked ATP release is significantly elevated in FIC cells. In both normal and FIC cells, ATP release was significantly decreased (mean 70% decrease) by application of blockers of stretch-activated channels (amiloride or gadolinium), as well as brefeldin A and monensin (mean 90% decrease), suggesting that ATP release occurs when ATP-containing vesicles fuse with the plasma membrane. Swelling-evoked release was reduced after removal of external calcium (65%), and release was blocked by incubation with BAPTA-AM or agents that interfere with internal calcium stores (caffeine, ryanodine, heparin, or 2-aminoethoxydiphenyl borate). In addition, agents known to act through inositol 1,4,5-triphosphate (IP3) receptors (thapsigargin, acetylcholine) release significantly more ATP in FIC compared with normal urothelium. Taken together, these results suggest that FIC results in a novel hypersensitivity to mechanical stimuli that may involve alterations in IP3-sensitive pathways.

Adenosine Triphosphate↗
PubMed2003

Nitric oxide synthase gene transfer for erectile dysfunction in a rat model.

OBJECTIVE: To determine whether over-expression of nitric oxide synthase (NOS) in the corpus cavernosum of the penis improves erectile function, as NO is an important transmitter for genitourinary tract function, mediating smooth muscle relaxation and being essential for penile erection. MATERIALS AND METHODS: The inducible form of the enzyme NOS (iNOS) was introduced into the corpus cavernosum of adult Sprague-Dawley rats (250-300 g) by injecting a solution of plasmid, adenovirus or adenovirus-transduced myoblast cells (adeno-myoblasts). Plasmid, adenovirus and adeno-myoblasts encoding the expression of the beta-galactosidase reporter gene were also injected into rats. RESULTS: Throughout the corpora cavernosum there was expression of beta-galactosidase after injecting each of the three solutions. Maximum staining was greatest for adeno-myoblast, then adenovirus and then plasmid. The mean (sd) basal intracavernosal pressure (ICP) of iNOS-treated animals (adenovirus and adeno-myoblast) increased to 55 (23) cmH2O, compared with naive animals with a basal ICP of 5 (6) cmH2O (P = 0.001). Stimulating the cavernosal nerve (15 Hz, 1.5 ms, 10-40 V, 1 min) resulted in a doubling of the ICP (adenovirus and adeno-myoblast) from the basal level of the iNOS-treated animals. Direct in situ measurement of NO showed the release of 1-1.3 micro mol/L in the adeno-myoblast penis. CONCLUSION: Myoblast-mediated gene therapy was more successful for delivering iNOS into the corpus cavernosum than direct adenovirus injection or plasmid transfection. Surprisingly, implanting muscle cells into the penis is not only feasible but also beneficial. Gene therapy for NOS may open new avenues of treatment for erectile dysfunction. Control of NOS expression would be necessary to prevent priapism.

Adenoviridae↗
PubMed2002

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1.

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1(-/-) mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1(-/-) mice, as was hypoosmolality-evoked ATP release from cultured trpv1(-/-) urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.

Acetic Acid↗
PubMed2001

Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection.

Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca(2+)-dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophin-deficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wild-type cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.

Animals↗
PubMed2001

Nitric oxide synthase gene therapy for erectile dysfunction: comparison of plasmid, adenovirus, and adenovirus-transduced myoblast vectors.

BACKGROUND AND PURPOSE: Nitric oxide (NO) has been recognized as an important transmitter for genitourinary tract function. This transmitter mediates smooth muscle relaxation and is essential for erection. The objective of our research was to determine whether overexpression of nitric oxide synthase (NOS) in the corpus cavernosum of the penis would correct erectile dysfunction. MATERIALS AND METHODS: We introduced the inducible form of the enzyme NOS (iNOS) into the corpus cavernosum of adult (250-300 g) male Sprague-Dawley rats by injecting a solution of plasmid, adenovirus, or adenovirus-transduced myoblast cells (adeno-myoblast) (N = 3-5 each group). We also injected plasmid, adenovirus, and adeno-myoblast encoding the expression of the beta-gatactosidase reporter gene. RESULTS: We noted expression of beta-galactosidase throughout the corpora cavernosum after injection of each of the three solutions. Staining was greatest for adeno-myoblast followed by adenovirus and then plasmid. The basal intracavernous pressure (ICP) of iNOS-treated animals (adenovirus and adenovirus-transduced myoblast) increased to 55 +/- 23 cm H(2)O v 5 +/- 6 H(2)O in naive animals (P = 0.001). Stimulation of the cavernous nerve (15 Hz, 1.5 msec, 10-40 V, 1 min) resulted in a twofold increase in ICP (adenovirus and adeno-myoblast) from the basal level of the iNOS-treated animals. Direct in situ measurement of NO demonstrated release of 1 to 1.3 microM NO in the adeno-myoblast-treated penis. CONCLUSION: Myoblast-mediated gene therapy was more successful in delivering iNOS into the corpus cavernosum than were the direct adenovirus or plasmid transfection methods. Gene therapy of NOS may open new avenues of treatment for erectile dysfunction. Control of NOS expression would be necessary to prevent priapism.

Adenoviridae↗
PubMed2000

Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein.

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca(2+) act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT(-/-)) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.

Animals↗
PubMed1998

Hypoxia and hypothermia enhance spatial heterogeneities of repolarization in guinea pig hearts: analysis of spatial autocorrelation of optically recorded action potential durations.

INTRODUCTION: Regional dispersions of repolarization (DOR) are arrhythmogenic perturbations that are closely associated with reentry. However, the characteristics of DOR have not been well defined or adequately analyzed because previous algorithms did not take into account spatial heterogeneities of action potential durations (APDs). Earlier simulations proposed that pathologic conditions enhance DOR by decreasing electrical coupling between cells, thereby unmasking differences in cellular repolarization between neighboring cells. Optical mapping indicated that gradients of APD and DOR are associated with fiber structure and are largely independent of activation. We developed an approach to quantitatively characterize APD gradients and DOR to determine how they are influenced by tissue anisotropy and cell coupling during diverse arrhythmogenic insults such as hypoxia and hypothermia. METHODS AND RESULTS: Voltage-sensitive dyes were used to map APs from 124 sites on the epicardium of Langendorff-perfused guinea pig hearts during (1) cycles of hypoxia and reoxygenation and (2) after 30 minutes of hypothermia (32 degrees to 25 degrees C). We introduce an approach to quantitate DOR by analyzing two-dimensional spatial autocorrelation of APDs along directions perpendicular and parallel to the longitudinal axis of epicardial fibers. A spatial correlation length L was derived as a statistical measure of DOR. It corresponds to the distance over which APDs had comparable values, where L is inversely related to DOR. Hypoxia (30 min) caused a negligible decrease in longitudinal thetaL (from 0.530 +/- 0.138 to 0.478 +/- 0.052 m/sec) and transverse thetaT (from 0.225 +/- 0.034 to 0.204 +/- 0.021 m/sec) conduction velocities and did not alter thetaL/thetaT or activation patterns. In paced hearts (cycle length [CL] = 300 msec), hypoxia decreased APDs (123 +/- 18.2 to 46 +/- 0.6 msec; P < 0.001) within 10 to 15 minutes and enhanced DOR, as indicated by reductions of L from 1.8 +/- 0.9 to 1.1 +/- 0.5 mm (P < 0.005). Hypothermia caused marked reductions of thetaL (0.53 +/- 0.138 to 0.298 +/- 0.104 m/sec) and thetaT (0.225 +/- 0.034 to 0.138 +/- 0.027 m/sec), increased APDs (128 +/- 4.4 to 148 +/- 14.5 msec), and reduced L from 2.0 +/- 0.3 to 1.3 +/- 0.6 mm (P < 0.05). L decreased with increased time of hypoxia and recovered upon reoxygenation. Hypoxia and hypothermia reduced L measured along the longitudinal (L(L)) and transverse (L(T)) axes of cardiac fibers while the ratio of L(L)/L(T) remained constant. CONCLUSION: Conventional indexes of DOR (i.e., APD "range" or "standard deviation," evaluated with extracellular electrodes) did not convey the spatial inhomogeneities of repolarization revealed by L. Spatial autocorrelation analysis provides a statistically significant measurement of DOR, which can take into account intrinsic heterogeneities of APDs and fiber orientation. The data show that hypoxia and hypothermia produce reductions of L, even though they have different effects on mean APD and conduction velocity. The preservation of a constant L(L)/L(T) ratio during hypoxia and hypothermia, despite large reductions in L, is consistent with a mechanism in which reduced cell-to-cell coupling unmasks intrinsic dispersions of APD and reduces L(L) and L(T) by the same factor. Thus, the spatial autocorrelation of APDs provides a sensitive index of DOR under normal and arrhythmogenic conditions. It incorporates the anisotropic nature of the myocardium and therefore is preferable to conventional indexes of DOR.

Action Potentials↗
PubMed1998

Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder.

Nitric oxide (NO) has been implicated in the regulation of the lower urinary tract. However, the source(s) of NO production in the urinary bladder (UB) has not been determined. Accordingly, we used a porphyrinic microsensor placed on the surface of UB strips in vitro to directly measure endogenous NO production. The afferent neurotoxin, capsaicin, and the mixed alpha/beta-adrenergic agonist, norepinephrine (NE), both evoked transient (1-3 s) NO release (range 50 nM to 1.4 microM). Adrenergic-mediated release was not decreased following denervation of the UB but was abolished following selective removal of the mucosa. On the other hand, release evoked by capsaicin (range 50-900 nM) was significantly decreased after UB denervation. These data indicate that NE releases NO from UB epithelium, and capsaicin releases NO from epithelium as well as nervous tissue in the UB. In light of reports that NO may regulate epithelial integrity and function in other tissues, agonist regulation of a constitutive nitric oxide synthase activity in the UB may provide a novel mechanism for modulation of bladder and urothelial function.

Adrenergic Agonists↗
PubMed1997

DMSO: effect on bladder afferent neurons and nitric oxide release.

PURPOSE: Interstitial cystitis (IC), a chronic disorder of the urinary bladder, is characterized by increased voiding frequency, urgency and pain. Patients with IC also exhibit reduced urinary nitric oxide synthase activity. Intravesical administration of dimethyl sulfoxide (DMSO) has been used to provide symptomatic relief in patients with IC. The present experiments were undertaken to determine if intravesical DMSO affects neural pathways involved in bladder function in the rat and if DMSO can influence the release of nitric oxide in the bladder or from afferent neurons. MATERIALS AND METHODS: The effects of intravesical DMSO (10% solution in saline) on reflex bladder activity, firing on bladder nerves and c-fos gene expression in spinal neurons was examined in urethane anesthetized female Wistar rats. The effect of DMSO (1-10%) on nitric oxide release from urinary bladder strips or acutely dissociated dorsal root ganglion cells was monitored in vitro with a porphyrinic microsensor. RESULTS: DMSO acutely increased reflex firing of pelvic nerve efferent axons, decreased bladder capacity and also increased neuronal c-fos expression in spinal cord regions that exhibit c-fos expression after chemical activation of capsaicin-sensitive bladder afferents. DMSO, like capsaicin, also directly released nitric oxide (NO) from both dissociated dorsal root ganglion neurons and from isolated strips of urinary bladder. CONCLUSIONS: These results suggest that DMSO induced stimulation of bladder afferent pathways and NO release from afferent neurons may be a reflection of the initial event in the desensitization of nociceptive pathways in the lower urinary tract (LUT).

Administration, Intravesical↗
PubMed1997

Beta-adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes.

Nitric oxide (NO) has been implicated in endogenous control of myocardial contractility. However, NO release has not yet been demonstrated in cardiac myocytes. Accordingly, endogenous NO production was measured with a porphyrinic microsensor positioned on the surface of individual neonatal or adult rat ventricular myocytes (n > 6 neonatal and adult cells per experiment). In beating neonatal myocytes, there was no detectable spontaneous NO release with each contraction. However, norepinephrine (NE; 0.25-1 microM) elicited transient NO release from beating neonatal (149 +/- 11 to 767 +/- 83 nM NO) and noncontracting adult (157 +/- 13 to 791 +/- 89 nM NO) cells. NO was released by adrenergic agonists with the following rank order of potency: isoproterenol (beta1beta2) > NE (alpha/beta1) > dobutamine (beta1) approximately epinephrine (alpha/beta1beta2) > tertbutylene (beta2); NO was not released by phenylephrine (alpha). NE-evoked NO release was reversibly blocked by N(G)-monomethyl-L-arginine, trifluoperazine, guanosine 5'-O-(2-thiodiphosphate), and nifedipine but was enhanced by 3-isobutyl-1-methylxanthine (0.5 mM = 14.5 +/- 1.6%) and BAY K 8644 (10 microM = 11.9 +/- 1%). NO was also released by A-23187 (10 microM = 884 +/- 88 nM NO), guanosine 5'-O-(3-thiotriphosphate) (1 microM = 334 +/- 56 nM NO), and dibutyryl adenosine 3',5'-cyclic monophosphate (10-100 microM = 35 +/- 9 to 284 +/- 49 nM NO) but not by ATP, bradykinin, carbachol, 8-bromoguanosine 3',5'-cyclic monophosphate, or shear stress. This first functional demonstration of a constitutive NO synthase in cardiac myocytes suggests its regulation by a beta-adrenergic signaling pathway and may provide a novel mechanism for the coronary artery vasodilatation and enhanced diastolic relaxation observed with adrenergic stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Shear stress induces ATP-independent transient nitric oxide release from vascular endothelial cells, measured directly with a porphyrinic microsensor.

Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca2+]i and NO release due to shear stress or agonist (ATP or brominated Ca2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 microL). The microsensor was positioned approximately 100 microns above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm2. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca2+]i, which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133 +/- 9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5 +/- 2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 mumol/L) or Br-A23187 (10 mumol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 mumol/L) chelation of extracellular Ca2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23 +/- 0.07 nmol/L per second) closely resembled the kinetics of Ca2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64 +/- 0.32 nmol/L per second). These data argue that shear stress causes a Ca(2+)-mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗