Regulation of cell volume and diffusion of intracellular water in salivary acinar cells.

The regulation of acinar cell volume and the properties of intracellular water were investigated in perfused rat mandibular salivary glands by proton nuclear magnetic resonance (NMR) spectroscopy. Using an inversion-recovery pulse sequence, and an extracellular relaxation reagent (10 mM Gd-DTPA) to suppress the proton NMR signal from extracellular water, acinar cell volume (intracellular water content) in unstimulated glands was shown to depend upon Cl- uptake by basolateral Na+-K+-2Cl- cotransport. Muscarinic and beta-adrenoceptor stimulation induced shrinkage and swelling respectively. In pulsed-field-gradient NMR experiments, the diffusion coefficient of intracellular water was found to be more than an order of magnitude smaller than that of extracellular water. Using this intrinsic difference in diffusivity between the two compartments, cell volume regulation was investigated in intact, perfused glands in the absence of relaxation reagents. Using both NMR techniques, acinar cells in perfused glands were observed to behave like simple osmometers in response to anisosmotic media, and did not show the volume regulatory responses described in dissociated acinar cells.

The in vivo relationship between blood flow, contractions, pH and metabolites in the rat uterus.

Little is known about the relationship between smooth muscle contractile activity and its blood supply. We have therefore investigated this in the rat uterus, using laser-Doppler flow measurement and intra-uterine pressure recordings. We found an inverse linear relationship between flow and contractile activity. There was no evidence for a critical level of flow, above which function is maintained and below which it declines; even small reductions in blood flow decreased uterine force. Force was rapidly restored upon reperfusion. Reactive hyperaemia was absent from all but 6 of the 41 preparations studied. We used 31P nuclear magnetic resonance (NMR) spectroscopy to measure concentrations of adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi) and intracellular pH (pHi) simultaneously with force and flow. Reductions in flow were associated with significant reductions in [ATP], [PCr] and pHi, and an increase in [Pi]. These changes were related to flow significantly and linearly and their effects on force may be additive. These data show that uterine smooth muscle is closely dependent upon its blood supply for maintaining both normal force production and metabolite levels. Consequently, even small decrements in flow may have deleterious functional effects.

Modulation of Na(+)-H+ exchange by altered cell volume in perfused rat mandibular salivary gland.

1. Intracellular pH (pHi) was measured by spectrofluorometry in perfused mandibular salivary glands isolated from the rat and loaded with the pH-sensitive fluoroprobe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell volume changes were estimated from changes in intracellular water content measured by proton NMR spectroscopy. 2. Stimulation with 1 microM acetylcholine (ACh) led to a 15 +/- 2% decrease in cell volume. A transient decrease in pHi was followed by a sustained increase (0.17 +/- 0.03 pH units) that has previously been attributed to the upregulation of the Na(+)-H+ exchanger. 3. Increasing perfusate osmolarity by addition of 60 mM sucrose caused a 19 +/- 2% decrease in cell volume and a sustained increase in pHi (0.12 +/- 0.01 pH units) that was abolished by 1 mM amiloride. Acid loading experiments indicated that the increase in pHi was due to an alkaline shift in the pH dependence of the Na(+)-H+ exchanger. 4. A 20% reduction in perfusate osmolarity prevented the cell shrinkage normally associated with ACh stimulation and largely abolished the ACh-induced increase in pHi. 5. Steady-state Na(+)-H+ exchanger activity, estimated from the initial rate of change in pHi following addition of amiloride, increased 9-fold during stimulation with ACh. When cell shrinkage was prevented by simultaneous exposure to the hypotonic solution, the activity of the exchanger still increased 7-fold in response to ACh. 6. We conclude that, although cell shrinkage leads to upregulation of the Na(+)-H+ exchanger, this factor alone is insufficient to account for the marked increase in exchanger activity that follows muscarinic stimulation.

A 31P NMR investigation into the effects of repeated vascular occlusion on uterine metabolites, intracellular pH and force, in vivo.

Little is known about the metabolic effects of ischaemia on high energy phosphates in vivo in smooth muscle. We have developed a method for reversibly occluding the uterine artery, which allows simultaneous measurement of uterine metabolites using 31P NMR spectroscopy, and intra-uterine pressure, in vivo during ischaemia. We have investigated the effects of repeated ischaemia on metabolites, intracellular pH and contractions in anaesthetized rats. Occlusion produced an immediate drop in uterine blood flow and decreased contractions. Although contractions recovered upon reperfusion after both occlusions, the contractile activity was less after the second period of occlusion, suggesting less resistance after a prior ischaemic period. Significant falls in [ATP] and [phosphocreatine] and an increase in [P(i)] occurred during both occlusions. These were all reversed within 30 min of reperfusion. There was a large drop in intracellular pH produced by occlusion, which was rapidly reversed upon reperfusion. The changes in metabolites and intracellular pH were similar during the repeated ischaemic period, to those occurring during the first ischaemic period suggesting no alteration in energy production or utilization had occurred, with prior exposure to ischaemia. The significance of these results to the functioning of the uterus in labour is briefly discussed.

An in vivo study of the effects of ischaemia on uterine contraction, intracellular pH and metabolites in the rat.

There are no data concerning the functional or metabolic effects of hypoxia in vivo in smooth muscle. We have therefore used 31P-NMR spectroscopy and intra-uterine pressure measurements to examine simultaneously, in vivo, the effect of ischaemia on uterine metabolites, intracellular pH (pHi) and force. A 1-2 cm portion of uterus from day 1 postpartum anaesthetized rats was exteriorized and an NMR surface coil placed on it. A balloon catheter in the uterine lumen recorded intra-uterine pressure changes from the same area. Reversible occluders were placed around the uterine artery. Occlusion produced a decrease and then abolition of contractions, within 10 min. In four of five animals contraction was abolished within 2 min. Upon reperfusion force was rapidly restored (1 min), in all preparations. The mean level of force was significantly above control (pre-occlusion) 20-30 min after reperfusion. The NMR data showed a significant fall in [ATP] (28%) and [phosphocreatine] (34%) during occlusion. Inorganic phosphate doubled in concentration during this period. Metabolites recovered slowly upon reperfusion, taking 20-30 min to return to pre-occlusion levels. The mean pHi fell from 7.32 to 7.00 upon occlusion and was rapidly reversed upon reperfusion. The changes in pHi closely correlated with the changes in uterine force. Decreases of pHi of a similar magnitude in vitro have previously been shown to abolish contractions; thus it is suggested that during ischaemia in vivo the depression of contraction is caused by the large fall in pHi.

Continuous measurement of cell volume changes in perfused rat salivary glands by proton NMR.

Changes in intracellular and extracellular water content have been measured in perfused rat salivary glands by repetitive application of an inversion recovery (IR) pulse sequence. The relaxation reagent Gd-DTPA (10 mM) was included in the perfusate so that the intracellular and extracellular water proton signals could be distinguished by their different longitudinal relaxation times. Changes in water content in response to altered perfusion pressure and perfusate osmolarity were determined at 30-s intervals and indicated a clear separation of the intracellular and extracellular components. Using a modification of the IR pulse sequence, changes in intracellular water content were also measured at 6-s intervals. With this time resolution, differences in the rates of cell shrinkage in response to hyperosmotic perfusates and the secretomotor agonist acetylcholine were observed. The results suggest that this approach offers a relatively noninvasive method for studying cell volume regulation in intact, perfused tissues and organs.

Continuous fluorometric measurement of intracellular pH and Ca2+ in perfused salivary gland and pancreas.

Intracellular pH (pHi) has been measured in intact, perfused rat mandibular salivary glands loaded with the fluorescent pH indicator BCECF [2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]. Glands mounted in the cuvette of a conventional bench-top spectro-fluorometer were perfused for 5 min with the acetoxymethyl ester of BCECF and fluorescence was measured ratiometrically at 6-s intervals. The mean value of pHi in glands perfused with a HCO3(-)-free, N-2-hydroxyethylpiperazine-N'-2- ethanesulphonic acid (HEPES)-buffered solution at 37 degrees C was 7.36 +/- 0.01 (n = 52) which is comparable with values obtained by 31P nuclear magnetic resonance (NMR) spectroscopy. NMR data confirmed that the BCECF loading period was accompanied by a transient acidification of the cells, but there was no significant change in the content of the major phosphorus metabolites. Changes in pHi in response to NH4Cl pulses and acetylcholine stimulation were comparable with results reported previously for isolated acini. Additional, preliminary experiments show that the method can also be used to monitor intracellular Ca2+ (using fura-2) in perfused salivary glands, and can be adapted for studies of the isolated, perfused pancreas.

Quantitative MRI of the prostate and uterus in monkeys.

Quantitative MRI has been carried out in the prostate, seminal vesicles, uterus, and ovaries in the pig-tailed monkey, Macaca nemestrina. T2-weighted, fat-suppressed, multislice experiments were performed at 2.35 T. Eight males, 14 ovariectomized females, and 20 intact females were studied. In the prostate, the caudal and cranial lobe were readily distinguished since the latter had a longer T2 value. For all tissues and organs, interanimal variations were large (up to 12-fold variation in volume), but reproducibility was excellent in the prostate and in the ovariectomized monkey uterus with coefficients of variation (CV) of 3 and 5%, respectively. In the intact monkey uterus, cycle-cycle reproducibility was good with CVs of 6-10% in the myometrium and 14-18% in the endometrium. In the follicular phase, endometrial growth (+3.8% day-1, P < 0.001) was accompanied by myometrial shrinkage (-1.6% day-1, P < 0.001), while in the luteal phase, growth was seen in both tissues (+4.3% day-1, P < 0.001 and +1.4% day-1, P < 0.001, respectively). The great value of these MRI techniques in obtaining data in pharmacological efficacy studies of endocrine drugs, and in limiting the number of animals used, is discussed.

Deuterium nuclear magnetic resonance imaging of tracer distribution in D2O clearance measurements of tumor blood flow in mice.

Proper implementation of direct injection clearance techniques to measure tumor blood flow (TBF) requires knowledge of the tracer distribution because TBF distribution is often inhomogeneous. Therefore, deuterium nuclear magnetic resonance imaging was used to follow tracer (HOD) distribution after direct injection of 10-40 microliters isotonic saline/D2O into RIF-1 tumors. Within 2 to 4 min after intratumor injection, tracer clearance was imaged by obtaining deuterium images every 1.4 min. The mean volume occupied by HOD in tumors in the first image acquired after injection with 10, 20, or 40 microliters D2O was 56 +/- 37 (SD) mm3, 44 +/- 2.9 mm3, and 174 +/- 83 mm3, respectively (n = 3 for each). In these control tumors, HOD was cleared from that volume without an appreciable increase in tracer distribution. In tumors heated for 45 min at 45 degrees C to greatly reduce TBF, the mean tracer volume in the first image after 10-microliters D2O injection was 41 +/- 10 mm3 and increased to 111 +/- 24 mm3 at 30 min (n = 3). For 10 microliters D2O injected at two distinct sites, the intensity decreased at each site while the sites remained separate (n = 6). The TBF at the two sites, measured independently by fitting the integrated HOD intensity from each site to a monoexponential decay function, was significantly different in only one of the six tumors examined. The use of deuterium nuclear magnetic resonance imaging to measure TBF from two (or more in larger tumors) independent sites provides a practical approach to assess TBF heterogeneity. The direct measurement of the tissue volume labeled with tracer and its dependence on injection volume should aid in determining how best to implement direct injection tracer clearance methods.