Surya M. Nauli

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca2+ influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP3 receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

Fibrocystin/Polyductin, Found in the Same Protein Complex with Polycystin-2, Regulates Calcium Responses in Kidney Epithelia

ABSTRACT Recent evidence suggests that fibrocystin/polyductin (FPC), polycystin-1 (PC1), and polycystin-2 (PC2) are all localized at the plasma membrane and the primary cilium, where PC1 and PC2 contribute to fluid flow sensation and may function in the same mechanotransduction pathways. To further define the exact subcellular localization of FPC, the protein product encoded by the PKHD1 gene responsible for autosomal recessive polycystic kidney disease (PKD) in humans, and whether FPC has direct and/or indirect cross talk with PC2, which, in turn, is pivotal for the pathogenesis of autosomal dominant PKD, we performed double immunostaining and coimmunoprecipitation as well as a microfluorimetry study of kidney tubular epithelial cells. FPC and PC2 are found to completely or partially colocalize at the plasma membrane and the primary cilium and can be reciprocally coimmunoprecipitated. Although incomplete removal of FPC by small interfering RNA and antibody 803 to intracellular epitopes of FPC did not abolish flow-induced intracellular calcium responses, antibody 804 to extracellular epitopes of FPC blocked cellular calcium responses to flow stimulation. These findings suggest that FPC and polycystins share, at least in part, a common mechanotransduction pathway.

Effects of nitric oxide and GABA interaction within ventrolateral medulla on cardiovascular responses during static muscle contraction.

We hypothesized that nitric oxide (NO) has opposing roles in regulating cardiovascular responses within the rostral (RVLM) and caudal (CVLM) ventrolateral medulla by modulating release of gamma-aminobutyric acid (GABA). We have measured GABA concentrations within the RVLM and CVLM during increases in mean arterial pressure (MAP) and heart rate (HR) following a 2-min tibial nerve stimulation-evoked static muscle contraction before and after microdialysis of the NO precursor, L-arginine (1.0 microM), for 30 min, and after the NO inhibitor, L-NMMA (1.0 microM), for 30 min. In eight anesthetized rats, muscle contraction significantly increased MAP, HR and GABA levels within the RVLM area (from 0.53+/-0.09 to 1.22+/-0.10 ng/10 microl). Following microdialysis of L-arginine, muscle contraction augmented GABA levels (from 0.45+/-0.07 to 2.18+/-0.09 ng/10 microl) and attenuated changes in MAP and HR. Subsequent application of L-NMMA significantly decreased GABA levels (from 0.47+/-0.08 to 0.22+/-0.07 ng/10 microl) but potentiated MAP and HR responses to a muscle contraction. In contrast, muscle contraction significantly increased MAP and HR but decreased GABA concentrations within the CVLM (from 1.20+/-0.20 to 0.78+/-0.17 ng/10 microl). Following microdialysis of L-arginine, muscle contraction significantly attenuated GABA levels (from 1.34+/-0.19 to 0.33+/-0.10 ng/10 microl) and augmented changes in MAP and HR in response to muscle contraction. A subsequent microdialysis of L-NMMA into the CVLM reversed the effects of L-arginine. These results demonstrate that NO within the RVLM and CVLM differentially modulates cardiovascular responses during static muscle contraction and that NO influences exercise-induced cardiovascular responses by modulating GABA release within the ventrolateral medulla.

Cardiovascular responses and neurotransmitter changes following blockade of nNOS within the ventrolateral medulla during static muscle contraction.

Nitric oxide (NO) is synthesized from L-arginine through the activity of the synthetic enzyme, NO synthase (NOS). Previous studies have demonstrated the roles of the three isoforms of NOS, namely endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) in cardiovascular regulation. However, no investigation has been done to study their individual role in modulating cardiovascular responses during static skeletal muscle contraction. In this study, we determined the effects of microdialyzing a specific nNOS antagonist into the rostral (RVLM) and caudal ventrolateral medulla (CVLM) on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex using rats. We hypothesized that the NO modulation of the exercise pressor reflex was largely influenced by specific nNOS activity within the ventrolateral medulla. Bilateral microdialysis of a selective nNOS antagonist, 1-(2-trifluoromethylphenyl)-imidazole (1.0 microM), for 30 or 60 min into the RVLM potentiated cardiovascular responses and glutamate release during a static muscle contraction. Levels of GABA within the RVLM were decreased. The cardiovascular responses and neurochemical changes to muscle contraction recovered following discontinuation of the drug. In contrast, bilateral application of the nNOS antagonist into CVLM attenuated cardiovascular responses and glutamate release during a static muscle contraction, but augmented GABA release. These results demonstrate that nNOS in the ventrolateral medulla plays an important role in modulating glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex, and contributes to the sympathoexcitatory and sympathoinhibitory actions of NO within the RVLM and CVLM, respectively.

Simultaneous glutamate and γ-aminobutyric acid release within ventrolateral medulla during skeletal muscle contraction in intact and barodenervated rats

The purpose of this study was to determine if baroreflex modulates cardiovascular responses and neurotransmitter release within rostral (RVLM) and caudal (CVLM) ventrolateral medulla during static contraction of skeletal muscle using anesthetized rats. We evoked cardiovascular responses by a static muscle contraction and measured simultaneous release of glutamate and gamma-aminobutyric acid (GABA) in both the RVLM and CVLM using microdialysis probes, two inserted bilaterally into the RVLM and two into the CVLM. In intact anesthetized rats, a muscle contraction increased release of glutamate concomitantly in both the RVLM and CVLM along with significant increases in heart rate and arterial blood pressure. In contrast, concentrations of GABA increased within the RVLM, but decreased significantly within the CVLM during the pressor response. These changes were due to contraction-evoked activation of muscle afferents since tibial nerve stimulation following muscle paralysis failed to evoke glutamate, GABA, or any cardiovascular changes. On the other hand, static muscle contractions in baroreceptor denervated rats augmented the increases in heart rate and blood pressure. Furthermore, muscle contraction significantly enhanced the release of glutamate in the RVLM but attenuated its release in the CVLM. In addition, concentrations of GABA within the RVLM were attenuated following a muscle contraction in denervated rats without any changes in GABA within the CVLM. These results demonstrate that the baroreceptors influence cardiovascular responses to static muscle contraction associated with dynamic changes in glutamate and GABA release within the RVLM and CVLM.

Cardiovascular responses and neurotransmission in the ventrolateral medulla during skeletal muscle contraction following transient middle cerebral artery occlusion and reperfusion.

We hypothesized that static skeletal muscle contraction-induced systemic cardiovascular responses, and central glutamate/GABA release in rostral (RVLM) and caudal ventrolateral medulla (CVLM), would be modulated by cerebral ischemia. In sham-operated rats, a 2-min tibial nerve stimulation induced static contraction of the triceps surae, evoked pressor responses, increased glutamate in both the RVLM and CVLM, decreased GABA in the CVLM, and increased GABA in the RVLM. In rats with a temporary 90-min left middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion, pressor responses during muscle contractions were attenuated, as were glutamate within the left RVLM and left CVLM. Glutamate within the right RVLM and right CVLM were unaltered and similar to those in sham rats. In contrast, GABA increases during muscle contractions were enhanced in the left RVLM and CVLM but changes within the right CVLM and RVLM were similar to those in sham rats. These results indicate that unilateral ischemia increases ipsilateral GABA/glutamate ratios during muscle contraction in the RVLM. In contrast, opposite changes in ipsilateral glutamate and GABA release within the RVLM and CVLM were observed following a 90-min right-sided MCAO followed by 24 h reperfusion. However, cardiovascular responses during muscle contraction were depressed following such an ischemic brain injury. These data suggest that transient ischemic brain injury attenuates cardiovascular responses to static exercise via modulating neurotransmission within the ventrolateral medulla.

Maturation alters the contribution of potassium channels to resting and 5HT-induced tone in small cerebral arteries of the sheep

To address the hypothesis that maturation alters the contribution of K-channels to resting and agonist-induced tone in small cerebral arteries, second branch middle cerebral arteries (approximately 200 microm) were taken from term fetal (139-141 days gestation) and adult sheep, denuded of endothelium, and mounted in myographs. After determination of length-tension relations, the arteries were stretched to 55, 100, and 145% of optimum length. At each level of stretch, contractile responses to 5 mM 4-aminopyridine (4-AP, voltage-sensitive K-channel blocker), 100 nM iberiotoxin (calcium-sensitive K-channel blocker), 10 microM glibenclamide (ATP-sensitive K-channel blocker), or 10 microM Ba(2+) (inward rectifier K-channel blocker) were recorded. In separate experiments, concentration--response relations were determined for 5-HT in the presence and absence of each of the four K-channel blockers at the same concentrations. Both 4-AP and iberiotoxin produced stretch-dependent contractions of greater magnitude in adult (37% for 4-AP and 43% for iberiotoxin at 100% optimum) than in fetal (5% for 4-AP and 7% for iberiotoxin at 100% optimum) arteries. 4-AP also enhanced the pD(2) for 5-HT in adult (from 7.15 to 7.49), but not in fetal, arteries. Conversely, glibenclamide attenuated the pD(2) for 5-HT in fetal (from 7.02 to 6.71), but not in adult, arteries. Iberiotoxin enhanced the pD(2) for 5-HT in both fetal (from 7.05 to 7.51) and adult (from 7.15 to 7.75) arteries. In addition, iberiotoxin enhanced maximum responses to 5-HT (from 59 to 82%) in adult but not fetal arteries. Finally, 4-AP enhanced the maximum responses to 5-HT in both fetal (from 67 to 85%) and adult (from 59 to 79%) arteries. These results indicate that maturation modulates the contribution of K(V), K(Ca), and K(ATP), but not K(IR) channels to basal and/or 5HT-induced cerebrovascular tone, and demonstrate that K(V) and K(Ca) channels are coupled to stretch-sensitive receptors, and that K(V) and K(Ca) limit contractile responses to 5-HT. To the extent that changes in pD(2) values reflect changes in agonist--ligand interactions, the data also suggest that K(V), K(Ca), and K(ATP) channels may possibly influence ligand--receptor binding for 5-HT.

Maturation alters cyclic nucleotide and relaxation responses to nitric oxide donors in ovine cerebral arteries.

To examine the hypothesis that maturation modulates nitric oxide (NO)-induced relaxation in cerebral arteries, we quantified concentration-relaxation relations and the corresponding dynamic responses of guanosine 3′:5′-cyclic monophosphate (cGMP) and adenosine 3′:5′-cyclic monophosphate (cAMP) levels following administration of nitroglycerin and S-nitroso-N-acetyl-penicilamine (SNAP), an NO donor, in posterior communicating and middle cerebral arteries from newborn (3–7 days) and adult sheep. The results offer 5 main observations: (1) the efficacy and potency of NO donors were generally greater in newborn than in adult cerebral arteries; (2) rates of relaxation, and presumably rates of NO release, were faster for equimolar concentrations of SNAP than for nitroglycerin in both newborn and adult arteries; (3) basal concentrations were greater for cAMP than for cGMP, and both were greater in newborn than adult cerebral arteries; (4) in adult cerebral arteries, NO-induced increases in cGMP occurred faster but relaxation developed more slowly than in newborn cerebral arteries, and (5) responses to NO donors involved significant cross-reactivity between cGMP and cAMP, the characteristics of which were age, artery, and agent specific. From these results, we conclude that postnatal changes in reactivity to NO reflect corresponding changes in soluble guanylate cyclase activity and possible decreases in NO half-life. We also conclude that maturation slows the mechanisms mediating NO-induced relaxation, and that this effect is more pronounced in distal than in proximal cerebral arteries. The data also suggest that the rate-limiting step governing rates of response to NO is probably downstream from cGMP synthesis. From the basal cyclic nucleotide levels, we conclude that basal ratios of synthesis to hydrolysis were greater in fetal than adult arteries. Because NO increased both cGMP and cAMP, we speculate that Type III phosphodiesterase has a possible influence upon cerebrovascular responses to NO, and that this influence varies with postnatal age and artery type. Together, these findings emphasize that the cerebrovascular effects of NO are highly age dependent and artery specific, and should be carefully considered when administering NO therapeutically in the neonate.

Molecular changes in nNOS protein expression within the ventrolateral medulla following transient focal ischemia affect cardiovascular functions

The majority of human strokes involve an occlusion of the middle cerebral artery and subsequent damage to the brain tissues it perfuses. We have previously reported that reflex cardiovascular changes during a static muscle contraction are attenuated following transient middle cerebral artery occlusion (MCAO) and reperfusion [A. Ally, S.M. Nauli, T.J. Maher, Cardiovascular responses and neurotransmission in the ventrolateral medulla during skeletal muscle contraction following transient middle cerebral artery occlusion and reperfusion, Brain Res. 952 (2002) 176-187]. We hypothesized that the attenuation is a result of altered expression of neuronal nitric oxide synthase (nNOS) within the rostral (RVLM) and caudal ventrolateral medulla (CVLM). In this study, we have compared cardiovascular responses and nNOS protein expression within the four quadrants, i.e., left and right sides of both RVLM and CVLM in sham-operated rats (n = 10) and in rats with a temporary 90-min left-sided MCAO followed by 24 h reperfusion (n = 10). Increases in mean arterial pressure during a static muscle contraction were significantly attenuated in MCAO rats when compared to sham rats. The transient ischemia reduced nNOS expression within the ipsilateral RVLM quadrant compared to the contralateral RVLM or RVLM quadrants of control rats. In contrast, compared to sham rats and the right CVLM quadrant of MCAO rats, nNOS expression was significantly augmented in the ipsilateral CVLM in left-sided MCAO rats. These data suggest that the attenuation of cardiovascular responses during static muscle contraction in MCAO rats is partly due to a reduction in nNOS expression within the ipsilateral RVLM and an overexpression of nNOS abundance within the ipsilateral CVLM. Results demonstrate that nNOS expression within the medulla plays a significant role in mediating cardiovascular responses during static exercise in intact and pathophysiological conditions.

Maturation attenuates the effects of cGMP on contraction, [Ca2+]i and Ca2+ sensitivity in ovine basilar arteries.

The present study explores the hypothesis that age-related variations in cerebrovascular responses to vasodilators reflect corresponding age-dependent differences in the mechanisms coupling changes in cytosolic cGMP to vasorelaxation. The experiments focused on cGMPs ability to decrease either [Ca2+]i or myofilament Ca2+ sensitivity, because both effects can contribute to cGMP-induced vasodilation. Use of the cGMP analog 8-pCPT-cGMP minimized problems associated with limited cell permeation or cGMP hydrolysis. In fetal basilars contracted with 10 microM serotonin, the EC30 for 8-pCPT-cGMP-induced relaxation was 6 microM. In fura-2 loaded fetal basilars, pretreatment with 6 microM 8-pCPT-cGMP significantly depressed the sensitivity of [Ca2+]i to 5HT, and also myofilament sensitivity to calcium, but only in fetal arteries. In fetal basilar arteries contracted with 120 mM potassium, the EC30 for 8-pCPT-cGMP-induced relaxation was 25 microM. In fura-2 loaded ovine arteries, pretreatment with 25 microM 8-pCPT-cGMP had no effect on the ability of graded concentrations of potassium to elevate [Ca2+]i but reduced potassiums ability to induce contraction and attenuated myofilament calcium sensitivity; these latter effects were significant only in fetal arteries. In alpha-toxin permeabilized preparations, 25 microM 8-pCPT-cGMP significantly depressed both basal- and agonist-stimulated myofilament calcium sensitivity, only in fetal but not in adult basilars. Together, these results demonstrate that: (1) sensitivity to cGMP is greater in fetal than adult sheep arteries independent of method of contraction; (2) cGMP can reduce [Ca2+]i but only in agonist-contracted and not in potassium-contracted arteries; (3) and cGMP attenuates myofilament calcium sensitivity regardless of method of contraction. Overall, the data demonstrate that variations in the ability of cGMP to produce vasodilatation reflect age-, artery-, and agonist-dependent differences in the combination of mechanisms mediating responses to cGMP.