Application Of Acetazolamide Research Articles

Lithium-induced NDI: acetazolamide reduces polyuria but does not improve urine concentrating ability.

Lithium is the mainstay treatment for patients with bipolar disorder, but it generally causes nephrogenic diabetes insipidus (NDI), a disorder in which the renal urine concentrating ability has become vasopressin insensitive. Li-NDI is caused by lithium uptake by collecting duct principal cells and downregulation of aquaporin-2 (AQP2) water channels, which are essential for water uptake from tubular urine. Recently, we found that the prophylactic administration of acetazolamide to mice effectively attenuated Li-NDI. To evaluate whether acetazolamide might benefit lithium-treated patients, we administered acetazolamide to mice with established Li-NDI and six patients with a lithium-induced urinary concentrating defect. In mice, acetazolamide partially reversed lithium-induced polyuria and increased urine osmolality, which, however, did not coincide with increased AQP2 abundances. In patients, acetazolamide led to the withdrawal of two patients from the study due to side effects. In the four remaining patients acetazolamide did not lead to clinically relevant changes in maximal urine osmolality. Urine output was also not affected, although none of these patients demonstrated overt lithium-induced polyuria. In three out of four patients, acetazolamide treatment increased serum creatinine levels, indicating a decreased glomerular filtration rate (GFR). Strikingly, these three patients also showed a decrease in systemic blood pressure. All together, our data reveal that acetazolamide does not improve the urinary concentrating defect caused by lithium, but it lowers the GFR, likely explaining the reduced urine output in our mice and in a recently reported patient with lithium-induced polyuria. The reduced GFR in patients prone to chronic kidney disease development, however, warrants against application of acetazolamide in Li-NDI patients without long-term (pre)clinical studies.

Chloride dysregulation and inhibitory receptor blockade yield equivalent disinhibition of spinal neurons yet are differentially reversed by carbonic anhydrase blockade.

Synaptic inhibition plays a key role in processing somatosensory information. Blocking inhibition at the spinal level is sufficient to produce mechanical allodynia, and many neuropathic pain conditions are associated with reduced inhibition. Disinhibition of spinal neurons can arise through decreased GABAA/glycine receptor activation or through dysregulation of intracellular chloride. We hypothesized that these distinct disinhibitory mechanisms, despite all causing allodynia, are differentially susceptible to therapeutic intervention. Specifically, we predicted that reducing bicarbonate efflux by blocking carbonic anhydrase with acetazolamide (ACTZ) would counteract disinhibition caused by chloride dysregulation without affecting normal inhibition or disinhibition caused by GABAA/glycine receptor blockade. To test this, responses to innocuous tactile stimulation were recorded in vivo from rat superficial dorsal horn neurons before and after different forms of pharmacological disinhibition and again after application of ACTZ. Blocking GABAA or glycine receptors caused hyperresponsiveness equivalent to that caused by blocking the potassium chloride cotransporter KCC2, but, consistent with our predictions, only disinhibition caused by KCC2 blockade was counteracted by ACTZ. ACTZ did not alter responses of neurons with intact inhibition. As pathological downregulation of KCC2 is triggered by brain-derived neurotrophic factor, we also confirmed that ACTZ was effective against brain-derived neurotrophic factor-induced hyperresponsiveness. Our results argue that intrathecal ACTZ has antiallodynic effects only if allodynia arises through chloride dysregulation; therefore, behavioral evidence that ACTZ is antiallodynic in nerve-injured animals affirms the contribution of chloride dysregulation as a key pathological mechanism. Although different disinhibitory mechanisms are not mutually exclusive, these results demonstrate that their relative contribution dictates which specific therapies will be effective.

EComment. Spinal cord protection during thoracoabdominal aneurysm repair

The study ‘Novel application of acetazolamide to reduce cerebrospinal fluid production in patients undergoing thoracoabdominal aortic surgery’ by Jafarzadeh and associates [1] comes to cover a large practice gap in the prolepsis of spinal cord ischaemia in patients undergoing thoracoabdominal aortic aneurysm (TAAA) repair. Our practice so far is to protect the spinal cord during TAAA repair with a spinal drain that is kept for 48-72 h in the postoperative period. Strict guidelines are applied for the management of the drain for optimal protection. In addition, permissive hypothermia and liberal use of alpha-agonists in order to increase the mean arterial pressure during the postoperative period is the standard routine. Finally, recent studies have stressed that adding intrathecal papaverine to the neuroprotective protocol for descending thoracic aneurysm and TAAA repairs may enhance spinal cord perfusion and provide additional spinal cord protection [2]. We agree with the authors that in cases of contra-indication to the use of a spinal drain or the inability to insert it, the use of other agents that increase spinal cord perfusion is promising. There is an imperative need of a randomized trial, which will determine the efficacy of acetazolamide in the spinal cord protection during TAAA repair.

Effects of acetazolamide on transient K+ currents and action potentials in nodose ganglion neurons of adult rats.

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K+ current (IA) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4‐AP attenuated or blocked the CO2‐induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch‐clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT‐PCR of mRNA and the expression of voltage‐gated K+ (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the IA by approximately 27% and the additional application of 4‐AP (1 mM) further inhibited IA by 48%. The application of 0.1 μM α‐dendrotoxin (α‐DTX), a slow inactivating transient K+ current (ID) blocker, inhibited the baseline IA by approximately 27%, and the additional application of 1 mM AZ further decreased the IA by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage‐gated K+ channel proteins were present, and most (80–90%) of the four Kv channels immunoreactive neurons showed the co‐expression of carbonic anhydrase‐II (CA‐II) immunoreactivity. These results indicate that the application of AZ causes the reduction in IA via the inhibition of four voltage‐gated K+ channel (Kv) proteins without affecting ID.

Quantification of cerebral blood flow in nonhuman primates using arterial spin labeling and a two-compartment model

Noninvasive absolute quantification of cerebral blood flow (CBF) with high spatial resolution is still a challenging task. Arterial spin labeling (ASL) is a promising magnetic resonance imaging (MRI) method for accurate perfusion quantification. However, modeling of ASL data is far from being standardized and has not been investigated in great detail. In this study, two-compartment modeling of monkey ASL data in three physiological conditions (baseline, sensory activated and globally elevated CBF) is reported. Absolute perfusion and arterial transit times were derived for gray matter (GM) and white matter (WM) separately. The uncertainties of the model's result were determined by Monte Carlo simulations. The fitted CBF values for GM were 133 ml/min/100 ml at baseline condition, 165 ml/min/100 ml during visual stimulation and 234 ml/min/100 ml for globally elevated CBF after intravenous injection of acetazolamide. The ratio of GM to WM CBF was 2.5 at baseline and was found to decrease to 1.6 after application of acetazolamide. The corresponding arterial transit times decreased from 742 to 607 ms in GM and from 985 to 875 ms in WM. Monte Carlo simulations showed that absolute CBF values can be determined with an error of 11-15%, while the arterial transit time values have a coefficient of variation of 25-31%. With an alternative acquisition scheme, the precision of the arterial transit times can be improved significantly. The CBF values in the occipital lobe of the monkey brain quantified with ASL are higher than previously reported in positron emission tomography studies.

Low cerebrovascular reserve capacity in long-term follow-up after subarachnoid hemorrhage

Background Intradural arteries formerly in vasospasm after subarachnoid hemorrhage (SAH) show structural changes that result in arterial wall thickening and luminal narrowing. To evaluate if these changes lead to maldistribution of cerebral perfusion and reduced cerebrovascular reserve capacity (CVRC) in surviving patients, a long-term follow-up study of 18 adult patients after SAH was performed. Methods Eighteen patients were selected for the study, all had shown vasospasm after an early operation on a ruptured aneurysm, were in good neurological condition (GOS [Glasgow Outcome Score] 4 or 5 ), and had no residual infarcts. A technetium-99m-hexamethyl-propylenamine oxime (HMPAO) single-photon emission computed tomography was performed 15 to 73 months after SAH. To study CVRC, a second investigation after application of acetazolamide was performed 1 week later. Results Single-photon emission computed tomography showed areas of focally reduced HMPAO uptake predominantly in the hemisphere ipsilateral to the vessels more affected by posthemorrhagic vasospasm. The thalamus and the basal ganglia, the frontal lobe, and the temporal lobe were the regions most frequently showing reduced uptake. The individual change of HMPAO uptake after acetazolamide application ranged from −7% to 44% (mean, 17% ± 15%). Conclusions These results show a remarkable reduction of CVRC compared with findings in healthy individuals. Based on these new findings, further investigations focusing on CVRC in routine SAH follow-up are worth being considered.

Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K+-deficient rats.

Carbonic-anhydrase (CA) inhibitors are used in the treatment of hypokalaemic periodic paralysis (hypoPP) and related channelopathies but their mechanism of action is unknown. Patch-clamp experiments and molecular modeling investigations were performed to evaluate the mechanism of actions of CA inhibitors on skeletal muscle Ca2+-activated-K+ (BK) channel of K+-deficient rats used as animal model of hypoPP. CA inhibitors showing different degree of CA inhibition such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT), which lacks inhibitory effects on CA enzymes, were tested in vitro on BK channels. The application of ACTZ, BFT, ETX, and DCP to excised patches activated the BK channel with potency: ACTZ(DE50=7.3x10(-6)M)>BFT(DE50=5.93x10(-5)M)>ETX(DE50=1.17x10(-4)M)>>DCP. In contrast, MTZ and HCT failed to activate the BK channel. Molecular modeling studies showed that the capability of CA inhibitors to open the BK channel was related to the presence in their structures of an intra-molecular hydrogen bond with calculated inter-atomic distances ranging between 1.82 A degrees and 3.01 A degrees and of an aromatic ring poor of electrons. ACTZ, BFT, ETX, and DCP showed these pharmacofores, while MTZ and HCT did not. Our data indicate that the activation of BK channel is a property of CA inhibitors that interact with the channel subunit/s and that this effect is not related to their capability to inhibit the CA enzymes.

The role of cerebral blood flow and cerebrovascular reserve capacity in the diagnosis of chronic hydrocephalus--a PET-study on 60 patients.

To investigate the clinical value of cerebral blood flow (CBF) and cerebrovascular reserve capacity (CVR) in the management of chronic hydrocephalus. Global cortical CBF and CVR in 60 patients (66 +/- 12 yrs) with chronic hydrocephalus were investigated before, one week (7 d) and 7 months (7 m) after shunting by 15-O-H2O PET (Siemens ECAT 951/31) at rest and after application of acetazolamide (1 g). After 7 months, clinical outcome was assessed according to Stein and Langfitt and patients were classified into responder (n = 31) and non-responder (n = 29). Before Surgery, responder had lower blood flow values compared to non responder (36 +/- 8 vs. 41 +/- 11 ml/100 ml/min; p = 0.04), whereas CVR was not different between outcome groups (33 +/- 10 vs. 41 +/- 8%; p > 0.05). After shunting, CVR in non-responder decreased from 41 +/- 8% to 32 +/- 5% (7 d), whereas in responder significant increases (p < 0.02) to 55 +/- 46% (7 d) and 54 +/- 31% (7 m) were observed. Regarding early individual changes in CVR, the majority (12/18; 66%) of non-responder had marked decreases in CVR-levels (< 30%), whereas clinical responder considerably improved in CVR (> 30%) in half of patients (7/14). Measurement of cerebral blood flow in chronic hydrocephalus might substantially contribute to selection of shunt candidates and neurological sequels may be rather related to early regeneration of the hemodynamic reserve.

Neuropsychological sequels to changes in global cerebral blood flow and cerebrovascular reserve capacity after shunt treatment in chronic hydrocephalus--a quantitative PET-study.

To study relationship of neuropsychological deficits in chronic hydrocephalus before and after shunting with dynamics in cerebral blood flow. In 27 patients (65 +/- 13 yrs) with idiopathic chronic hydrocephalus 11 selected neuropsychological tests, providing a wide range of psychomotor functions, were performed before, one week (early) and 7 months (late) after shunting. Parallel global cortical blood flow (CBF) and cerebrovascular reserve capacity (CVR) were determined by dynamic 15-O-water PET studies (Siemens ECAT 951/31) before and after application of acetazolamide (1 g). Test raw data and changes after treatment were compared with global and regional blood flow values by polynomial regression. No relationship of test profiles with hemodynamics before surgery was found. After one week, improvement of gait was related to an increase in cerebrovascular reserve capacity (p = 0.05). After 7 months changes in mental tests were related to changes in hemodynamics: again increases in CVR were significantly related to improvements in visual attention and verbal memory (p < 0.01). Early improvement of hemodynamic reserve was related to improvement in gait, whereas mental improvement was particularly related to increases in global CVR at later periods after shunting. These results indicate that neurological sequels in idiopathic chronic hydrocephalus after shunting may actually depend on consecutive improvement of cerebral hemodynamics.

Upregulation of the hyperpolarization-activated cation current in rat thalamic relay neurones by acetazolamide.

1. The effect of inhibition of brain carbonic anhydrase (CA) on the hyperpolarization-activated cation current (Ih) of thalamocortical (TC) neurones of the rat ventrobasal thalamic complex (VB) was investigated in an in vitro slice preparation using the whole-cell patch-clamp technique and fluorescence ratio imaging of the pH indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. Recording of Ih before and after addition of 0.4-0.8 mM acetazolamide to the bathing fluid revealed a significant shift in the voltage dependence of activation (V ) of 5-7 mV to more positive potentials. 3. Simultaneous recording of Ih and BCECF fluorescence ratio (F420/F495) revealed an increase in Ih amplitude accompanied by an intracellular alkalinization upon application of acetazolamide. The CA inhibitor ethoxyzolamide (EZA, 50 microM) also led to an intracellular alkalinization and a subsequent 4-5 mV positive shift of V of Ih. 4. Acetazolamide and EZA both profoundly slowed the rapid fall of pHi upon switching from Hepes- to CO2/HCO3--buffered superfusate, indicating intracellular CA isoforms in TC neurones. 5. In slices bathed in Hepes-buffered saline, addition of acetazolamide had no effect on the amplitude and time course of activation of Ih, indicating that the action of acetazolamide on Ih was dependent on the presence of HCO3-. 6. Under current-clamp conditions, the neuronal response to hyperpolarizing current pulses in the presence of acetazolamide was decreased as compared to control. This resulted in a strongly reduced ability of TC neurones to produce rebound Ca2+-mediated spikes. 7. The present results implied that in TC neurones acetazolamide led to an intracellular alkalinization which causes, due to its pH sensitivity, an increase in Ih.

Neuroactive steroids induce GABA(A) receptor-mediated depolarizing postsynaptic potentials in hippocampal CA1 pyramidal cells of the rat.

Intracellular recordings were performed in area CA1 pyramidal cells of rat hippocampal slices to determine the effects of certain steroids on inhibitory postsynaptic potentials/currents (IPSP/Cs) mediated by GABA(A) receptors. Following application of the steroids 5alpha-pregnan-3alpha,21-diol-20-one (5alpha-THDOC), alphaxalone and 5beta-pregnan-3alpha-ol-20-one (pregnanolone) hyperpolarizing PSPs developed into biphasic responses consisting of an early hyperpolarizing and a late depolarizing PSP sequence. Steroid-induced depolarizing PSPs could be elicited in the presence of antagonists to non-NMDA, NMDA, and GABA(B) receptors, indicating that these receptor types do not contribute significantly to the initiation of these responses. Depolarizing PSPs were completely blocked by both GABA(A) receptor antagonists bicuculline and t-butylbicyclophosphorothionat (TBPS) providing evidence for their mediation by GABA(A) receptors. The reversal potential of steroid-induced late inward PSCs, measured in single-electrode voltage clamp, was -29.9+/-5.3 mV, whereas the early outward current, which corresponded to the early hyperpolarizing component of PSPs, reversed at -68.2+/-1.5 mV. Depolarizing PSPs and late inward PSCs were sensitive to reduction of extracellular [HCO3-] and block of carbonic anhydrase by application of acetazolamide. The results suggest that certain neuroactive steroids can induce GABA(A) receptor-mediated depolarizing PSPs, which are dependent on HCO3-.

Correlation of Transcranial Doppler Sonography Mean Flow Velocity with Cerebral Blood Flow in Patients with Intracranial Pathology

Several studies suggest that relative changes in cerebral blood flow (CBF) may be assessed via transcranial Doppler sonography (TCD). The present study investigates the correlation between changes in TCD-mean flow velocity (Vm) and changes in CBF in patients with a variety of types of intracranial pathology undergoing cerebrovascular reactivity tests. After informed consent was obtained, 32 patients presenting with stenoses of brain-supplying arteries (n = 13), cerebral vascular malformations (n = 6), surgical decompression for subarachnoid hemorrhage (n = 2), brain edema after closed head injury (n = 8), or hepatic encephalopathy (n = 3) were studied. The patients were divided into two groups for different reactivity tests. Patients in group 1 (awake or sedated, n = 18) received a 1-g dose of acetazolamide intravenously. In group 2 (n = 14), mechanical ventilation was adjusted to produce a 20% decrease in arterial CO2 tension compared with baseline. Regional CBF was measured using xenon-enhanced computed tomography (Xe-CT). Xe-CT scans at the levels of the basal ganglia and the lateral ventricles were performed during a 4.5-min xenon wash-in period. Bilateral flow velocity was measured in the middle cerebral artery using a 2-MHz pulsed TCD system. Mean arterial blood pressure, heart rate, and end-tidal CO2 were continuously recorded during the procedure. After baseline measurements and either alteration of CO2 or application of acetazolamide, the cerebrovascular reactivity was assessed at 20 min by a second measurement of CBF, TCD, and all other physiologic variables. The correlation coefficient for relative changes of MCA territory CBF versus Vm and for the overall population was r = 0.82. In groups 1 and 2, the r values were 0.39 and 0.5, respectively. Correlation coefficients did not exceed r = 0.4 in any subgroup-classification based on diagnosis. The close correlation between changes in CBF and Vm (r = 0.82) in patients with heterogeneous intracranial pathology seems to show that TCD is a measure of CBF. However, in groups 1 and 2 and in subgroups formed of patients classified according to diagnoses, data dispersion suggests that the actual correlation is weaker. Relation of changes in Vm to those in CBF may depend on the underlying diagnosis. These data indicate that the correlation between Vm and CBF may vary with intracranial pathology.

Assessment of cerebral blood flow reserve using functional magnetic resonance imaging.

Imaging of activated brain areas based on changes of blood deoxyhemoglobin levels is now possible with MRI. Acetazolamide (ACZ) increases cerebral blood flow (CBF) without changing cerebral oxygen consumption; this results in signal changes observed in gradient echo MR images from the areas with an increase in CBF. We assessed signal changes after ACZ application in seven healthy subjects with a conventional 1.5-T MRI scanner. The susceptibility-sensitized three-dimensional fast low-angle shot (FLASH) sequence was used to visualize signal changes induced by ACZ. We analyzed anatomic localization of different ranges of detected signal changes. ACZ caused significant signal changes in the gray matter and at the edge of the cerebral cortex, the latter corresponding to draining surface veins. No significant differences were seen among different brain areas within the same slice. Using the maximal intensity projection technique, we were able to partially separate signal changes originating in draining veins from signal originating in the gray matter microvasculature. Signal changes from the microvessels reflect cerebrovascular reserve. Blood-oxygen-level-dependent (BOLD) based MRI can evaluate CBF reserve with high spatial and temporal resolution. To assess cerebrovascular reserve, it is necessary to separate signal changes originating in large vessels from signal from brain microvasculature.

Effect of the application of acetazolamide soaked contact lenses on intraocular pressure of rabbits

Topical application of acetazolamide has no known effect on intraocular pressure (IOP). We tried to detect the hypotensive effect on IOP of acetazolamide soaked onto soft contact lenses (CL). We applied CLs soaked in either 1%, 3%, or 5% acetazolamide solution onto one eye of 29 rabbits while the contralateral eye served as a control. There was an average 32% reduction of IOP amongst all acetazolamide applied eyes, and an average 19% reduction of IOP amongst all control eyes. Amongst the 1% acetazolamide-CL applied eyes there was a mean 37% reduction of IOP, amongst the 3% acetazolamide-CL applied eyes a mean 36% reduction, amongst the 5% acetazolamide-CL applied a mean 30% reduction, and a mean 19% reduction in control eyes. The longest period of IOP reduction followed the application of 1% acetazolamide-CLs, probably owing to improved drug corneal penetration at this concentration. Our results reveal that the application of acetazolamide soaked soft CLs has a significant hypotensive effect on IOP in both the applied and contralateral control eyes of rabbits.

Mechanism of action of GABA on intracellular pH and on surface pH in crayfish muscle fibres.

1. The mode of action of gamma-aminobutyric acid (GABA) on intracellular pH (pHi) and surface pH (pHs) was studied in crayfish muscle fibres using H(+)-selective microelectrodes. The extracellular HCO3- concentration was varied (0-30 mM) at constant pH (7.4). 2. GABA (5 x 10(-6)-10(-3) M) produced a reversible fall in pHi which showed a dependence on the concentrations of both GABA and HCO3-. The fall in pHi was associated with a transient increase in pHs and it was inhibited by a K(+)-induced depolarization. 3. In the presence of 30 mM-HCO3-, a near-saturating concentration of GABA (0.5 mM) produced a mean fall in pHi of 0.43 units. This change in pHi accounted for about two-thirds of the GABA-induced decrease (from -66 to -29 mV) in the sarcolemmal H+ driving force, while the rest was due to the simultaneous depolarization. 4. The apparent net efflux of HCO3- (JHCO3e) produced by a given concentration of GABA was estimated on the basis of the instantaneous rate of change of pHi. In the presence of 30 mM-HCO3-, JHCO3e following exposure to 0.5 mM-GABA had a mean value of 8.0 mmol l-1 min-1. Under steady-state conditions (at plateau acidosis), the intracellular acid load produced by 0.5 mM-GABA was about 25% of that seen at the onset of the application. 5. The GABA-induced HCO3- permeability, calculated on the basis of the flux data, showed a concentration dependence similar to that of the GABA-activated conductance described in previous work. 6. The GABA-induced increase in pHs was immediately blocked by both a membrane-permeant inhibitor of carbonic anhydrase (acetazolamide, 10(-6) M) and by a poorly permeant inhibitor (benzolamide, 10(-6) M). 7. Application of acetazolamide (10(-4) M) for 5 min or more produced a decrease of up to 60% in the maximum rate of fall of pHi at GABA concentrations higher than 20 microM. 8. The recovery of the GABA-induced acidosis was associated with a fall in pHs. The recovery was completely blocked in solutions devoid of Na+ or of Cl-, as well as by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, 10(-5) M). This indicates that the maintenance of a non-equilibrium H+ gradient at plateau acidosis and the recovery of pHi are attributable to Na(+)-dependent Cl(-)-HCO3- exchange. 9. We conclude that the effects of GABA on pHi and pHs are due to electrodiffusion of HCO3- across postsynaptic anion channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiorespiratory effects induced by acetazolamide on the ventromedullary surface of the cat.

1. Inhibition of carbonic anhydrase by acetazolamide in alpha-chloralose-anaesthetized cats, in a region of the brain stem co-extensive with the glycine-sensitive area, intermediate chemosensitive area, and probably C1 catecholaminergic neurones produces hypotension, bradycardia and depression of the central respiratory drive. 2. These responses are concentration dependent, and can still be observed when the enzyme substrate (CO2) is elevated. Therefore, in both the hypercapnic and the normocapnic condition, similar responses in arterial blood pressure, heart rate and respiratory rate are observed when acetazolamide is topically applied to the glycine-sensitive area. 3. To investigate further the contribution of peripheral baro-, chemo- and cardiopulmonary receptors to these responses, acetazolamide was topically applied to the glycine-sensitive area under three different conditions: intact gallamine-paralysed (5 mg kg-1 h-1) and artificially ventilated (A), sinoaortic denervated (B), and sinoaortic denervated plus bilaterally vagotomized cats (C). Under all conditions, similar responses were observed. The fall in arterial blood pressure was 75 +/- 11 (A), 90 +/- 13 (B), and 75 +/- 9 mmHg (C). Changes in heart rate during acetazolamide application were -23 +/- 6, -20 +/- 8, and -26 +/- 6 beats min-1, respectively. The decreases in respiratory rate were 9 +/- 2 (A), 11 +/- 2 (B), and 11 +/- 2 breaths min-1 (C). 4. The data indicate that the responses to topical application of acetazolamide are mainly due to its central action at the glycine-sensitive area and are not influenced by peripheral baroreceptor and chemoreceptor inputs.

A microelectrode for continuous recording of volume fluxes in isolated perfused tubule segments.

Manufacture, properties and use of a micro enzyme electrode for continuous monitoring of volume fluxes in the isolated tubule preparation is described. The specific electrode is a galactose-oxidase enzyme electrode, which can be used to detect changes in raffinose concentrations. The electrode's response to raffinose is almost linear over concentrations from 0-12 mmol/l. The electrode equally responds to galactose as to raffinose but is insensitive to other sugars, to pH changes (from 6.0-8.0), CO2 (from 1-10%) and electrolytes tested. Reducing O2 from 100 to 10% and to 1%, leads to a reduction of the reading by 10% and 30%, respectively. The reading is almost doubled when the temperature is increased from 20-40 degrees C. Furthermore, reducing agents such as uric acid and ascorbic acid interfere with the reading. If these substances and raffinose are omitted from the perfusate for isolated perfused proximal mouse tubules, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce substances in sufficient amounts to interfere with the electrode reading. After addition of 6 mmol/l raffinose to the perfusate the raffinose concentration in the collected fluid of 0.76 +/- 0.05 mm segments of straight proximal mouse tubules (perfusion rate = 3.4 +/- 0.45 nl/min) is 10.2 +/- 0.3 mmol/l, indicating a volume reabsorption of 1.5 +/- 0.3 nl/min. Peritubular application of acetazolamide reduces the volume reabsorption by 42 +/- 4%.