Effects of osmotic pressure on prolactin and growth hormone secretion from organ-cultured eel pituitary

Summary Effects of medium osmotic pressure on the release of prolactin (PRL) and growth hormone (GH) from the pituitary of the Japanese eel, Anguilla japonica, were examined during long-term organ culture in a defined medium. Prolactin and GH release, as measured by homologous radioimmunoassays, increased gradually for 7 days during incubation in isosmotic medium (295 mOsmolal). On day 7, 3 to 5 times more PRL and GH were released than on day 1. The amount of GH released was about 100 times greater than that of PRL. Electron microscopic observation revealed that both PRL and GH cells were in good condition after 7 days incubation. The reduction of medium osmotic pressure from 295 (isosmotic) to 235 or 260 mOsmolal significantly stimulated PRL release for 4 days. By contrast, an increase in medium osmolality from 295 to 360 mOsmolal was without effect. These treatments produced no significant alterations in GH release. The stimulatory effect of hyposmotic medium (235 mOsmolal) was no longer evident by 12 h after the pituitaries were returned to isosmotic medium. The isosmotic but low-sodium medium, prepared by adding mannitol to the hyposmotic medium, did not stimulate PRL release from the pituitary. These results indicate that plasma osmolality may be an important physiological factor controlling PRL release during freshwater adaptation of the eel.Abbreviations GH growth hormone - OAPBS PBS with 1% ovalbumin - PAGE polyacrylamide gel electrophoresis - PBS phosphatebuffered saline - PRL prolactin - rER rough endoplasmic reticulum     

Direct hyposmotic stimulation of gastric acid secretion

Gastric glands isolated from rabbit stomach were incubated in isosmotic medium or media made hyposmotic by 50-100 mOsm/kg. As indicated by radiolabeled aminopyrine accumulation, acid secretion was nearly 3 times greater in 200 mOsm/kg hyposmotic than in isosmotic medium after a 30-min incubation. The hyposmotic stimulation appeared within 2 min, peaked at 10-15 min and declined almost to the isosmotic control by 45 min. As estimated by the wet weight corrected for inulin extracellular space, the intracellular water of the glands also peaked at 15 min and returned to the isosmotic norm by 45 min. Hyposmotic stimulation of acid secretion directly involved the parietal cell, since parietal cells obtained from gastric glands were also stimulated. That the hyposmotic response was direct was indicated by omeprazole inhibition of aminopyrine accumulation in hyposmotic medium.     

Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.     

Histamine-mediated hyposmotic stimulation of gastric acid secretion

Gastric glands incubated in hyposmotic medium (200 mOsm) accumulated aminopyrine, a measure of acid secretion, to the same extent as that of paired glands in isomotic medium containing histamine (10(-4) M). These maximal responses to hyposmolality and histamine were not additive. The hyposmotic response peaked earlier than the histamine response. Hyposmotic stimulation was nearly abolished by preincubation of the glands with metiamide and cimetidine, H-2 histamine antagonists. In the presence of histaminase, no hyposmotic stimulation occurred. The response to forskolin, a stimulant of adenylate cyclase, was equivalent in hyposmotic and isosmotic media. These results indicate that hyposmolality releases histamine from a paracrine cell in the gastric gland and that histamine binds to H-2 receptors on the parietal cell to initiate a cyclic AMP-mediated stimulation of acid secretion.     

PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT BUFFALO PROLACTIN

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

Secretion of prolactin and growth hormone by teleost pituitariesin vitro

Summary The osmotic pressure (OP) of the incubation medium directly affects synthesis and release of prolactin byPoecilia andAnguilla pituitariesin vitro.There is an inverse relationship between the OP of the medium and the amount of prolactin released into the medium (Pigs. 1, 2; Tables 1, 3).Release of newly-synthesised prolactin is similarly related to OP of the medium (Pigs. 3,4; Table 3).Histometric data of prolactin cells also suggests that hormone synthesis is reduced in high OP media (Table 2).Release and synthesis of putative growth hormone (GH) byPoecilia pituitaries is not influenced by OP of the medium (Table 4), butAnguilla pituitaries release more GH in low OP media (Table 5).We thank Mr. W. Thomson for technical assistance. One of us, P. M. I., receives financial support from the Yorkshire Cancer Research Campaign and the work was also supported in part by USPHS Grant AMI-3795. B. I. B. is Sorby Research Fellow of the Royal Society, London.     

Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K⁺ channel inhibitors Ba²⁺, quinine, and tetraethylammonium chloride, implicating K⁺ channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K⁺ channel, and also a dominant-negative NH₂-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K⁺ current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K⁺ secretion. potassium channel; volume regulation; mammary gland     

Osmotic and volume control of diuresis in conscious ducks (Anas platyrhynchos)

Summary In conscious Pekin ducks made diuretic either by infusing hyposmotic glucose solution or isosmotic saline, osmotic and volume effects on renal water excretion were investigated. As in mammals, antidiuresis mediated by enhanced release of antidiuretic hormone was induced by increasing carotid blood osmolality while a decrease augmented diuresis, indicating cerebral osmotic control of renal water excretion in birds.In contrast to the situation in mammals, a sensitive diuretic response to isosmotic volume expansion, corresponding to 1% of the extracellular volume, can be demonstrated, with intracarotid and intravenous application of the isosmotic saline infusion having identical effects.Volume loading with isosmotic saline produced a greater diuretic response than loading with the same amount of autologous blood, thereby indicating a major contribution of volume changes in the interstitial compartment to the control of renal water excretion. This corresponds to the importance of the interstitial fluid compartment for the control of salt gland activity in this species.Abbreviations AVP arginine vasopressin - ECF extracellular fluid - i.c., i.v. intracarotid, intravenous - ECFV ECF volume     

Effect of L-DOPA,Bromocripton and Lergotrile on prolactin cell activity and hydromineral regulation in yearling coho salmon,Oncorhynchus kisutch

Synopsis The prolactin inhibiting substances L-DOPA, Lergotrile and Bromocripton appeared to reduce the secretory activity of the prolactin cells in coho salmon (Oncorhynchus kisutch) yearlings acclimated to distilled water and effected significant reductions in plasma osmotic pressure and plasma Na⁺ and/or Cl^– concentrations. This suggests that in coho salmon prolactin is involved in osmotic or ionic homeostasis in hyposmotic environments in a manner similar to that in other teleosts.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

The Response of Prolactin,ACTH, and Growth Hormone Cells in the Pituitary Gland of the Three-Spined Stickleback,: Gasterosteus aculeatus L. form leiurus,to Increased Environmental Salinities

Benjamin, M. 1980. The response of prolactin, ACTH, and growth hormone cells in the pituitary gland of the three-spined stickleback, Gasterosteus aculeatus L. form leiurus, to increased environmental salinities. (Department of Anatomy, University College, Cardiff, Wales, U.K.) — Acta zool. (Stockh.) 61(1): 1–7. The time-sequence of response of the prolactin, ACTH and growth hormone cells in the pituitary gland of the leiurus form of the three-spined stickleback, Gasterosteus aculeatus L., to a transfer from freshwater to seawater, was studied by light microscopy. The appearance of the pituitary was compared with that of animals caught in brackish or sea water. The prolactin cells respond only slowly to seawater by cytological changes interpreted as signs of decreased secretory activity. It is thus suggested that prolactin is unlikely to be important for osmoregulation in this stickleback. The growth hormone cells are more active in all seawater-adapted animals, while the ACTH cells are less active—although only in laboratory experiments. The differing responses of the pituitaries of the leiurus and trachurus forms of G. aculeatus and Pungitius pungitius to high salinities are compared. Even in species whose pituitaries are virtually identical, the response to high salinites may differ widely.     

Effect of extracellular osmolality on cell volume and resting metabolism in mammalian skeletal muscle

The purpose of the present investigation was to establish an in vitro mammalian skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated, whole muscles [soleus and extensor digitorum longus (EDL)] were dissected from Long-Evans rats and incubated for 60 min in Sigma medium 199 (1 g of resting tension, bubbled with 95% O(2)-5% O(2), 30 +/- 2 degrees C, and pH 7.4). Medium osmolality was altered to simulate hyposmotic (190 +/- 10 mmol/kg) or hyperosmotic conditions (400 +/- 10 mmol/kg), whereas an isosmotic condition (290 +/- 10 mmol/kg) served as a control. After incubation, relative water content of the muscle decreased with hyperosmotic and increased with hyposmotic condition in both muscle types (P < 0.05). The cross-sectional area of soleus type I and type II fibers increased (P < 0.05) in hyposmotic, whereas hyperosmotic exposure led to no detectable changes. The EDL type II fiber area decreased in the hyperosmotic condition and increased after hyposmotic exposure, whereas no change was observed in EDL type I fibers. Furthermore, exposure to the hyperosmotic condition in both muscle types resulted in decreased muscle ATP and phosphocreatine (P < 0.05) contents and increased creatine and lactate contents (P < 0.05) compared with control and hyposmotic conditions. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acute alterations in muscle water content and resting muscle metabolism.     

Hyposmotic fluid formation in Hydra

A detailed model for hyposmotic fluid formation in Hydra is presented. We propose that enteron fluid formation occurs in two steps: (1) segregation of an isosmotic fluid in large intercellular vacuoles with (2) subsequent reabsorption of solute in the intercellular channels to form the hyposmotic fluid of the enteron. Intercellular spaces in Hydra have been studied by light microscopy and thin-section electron microscopy, as well as by electrophysiological methods. These spaces are of two types: (1) large vacuoles which are located in the cells of both the epidermis and gastrodermis, being more numerous in the epidermis; and (2) lateral intercellular channels which run from the intercellular vacuoles, leading eventually to the enteron. These vacuoles and channels are highly convoluted, forming a complex three-dimensional network. We suggest that this network is involved in the water balance of Hydra.     

Na+ channel activity in cultured renal (A6) epithelium: Regulation by solution osmolarity

Summary Solution osmolarity is known to affect Na⁺ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na⁺ currents (I _sc).I _sc for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25±2, 9±2, 3±0.4, and 0.6±0.5 A/cm², respectively. Similar results were obtained following changes in the serosal tonicity; mucosal changes did not significantly affectI _sc. The changes inI _sc were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na⁺ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na⁺ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na⁺ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na⁺ transport rates for A6 epithelia reported in the literature.     

Vascular smooth muscle contraction evoked by cell volume modulation: role of the cytoskeleton network.

Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca(2+) channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca(2+)-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca(2+) channel-mediated contraction of swollen vascular SMC and Ca(2+)(o)-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.     

Evidence for an inhibitory influence of rat placental lactogen on prolactin release in vitro

Incubation of placental tissue from Day 11 pregnant rats for increasing periods of time resulted in proportionately more rat placental lactogen (rPL) release. The amount of placental tissue incubated correlated directly with the amount of rPL released into the medium. When placentas were coincubated with anterior pituitaries from ovariectomized rats, prolactin release was significantly inhibited. When media from incubations which had contained varying numbers of Day 11 placentas for 24 h were added to vials containing anterior pituitaries, prolactin release was inhibited, proportionate to the amount of rPL in the media. Media from incubations of Day 9 placentas, which contained very little rPL, had no effect on prolactin release. When medium containing anterior pituitary tissue was incubated for 24 h, pituitaries removed, and the medium incubated with placental tissue for an additional 24 h, there was no difference in prolactin levels compared to incubation medium not containing placental tissue. Addition of a trypsin inhibitor to the medium containing placental tissue did not augment the amount of prolactin remaining after a 24-h incubation. Thus it would appear that the placenta does not release a substance into the medium that destroys prolactin. This suggests that secretions from the placenta, presumably rPL, can exert a negative feedback on prolactin secretion at the level of the anterior pituitary.     

The influence of prolactin (PRL) on progesterone secretion by porcine luteal cells in vitro

Porcine luteal cells were obtained from corpora lutea on the 5th, 13th and 17th days of the estrous cycle. The cells were suspended at a concentration of 5 × 10⁴ cells/ml in Eagle's medium with 2% human serum albumin. These cells were incubated with or without 0.01, 0.1, 1 or 10 μg/ml porcine prolactin. The amount of progesterone in cultures was estimated by a radio-immunological method after 30 min, 3 h and 6 h of culturing.Luteal cells obtained on the 5th day of the estrous cycle and incubated without prolactin secreted 71.24 ± 21.91 ng progesterone/ml of medium, whereas under the influence of prolactin at 0.01, 0.1, 1 and 10 μg/ml, 39.06 ± 13.33, 44.31 ± 12.69, 44.88 ± 16.85 and 51.62 ± 15.01 ng progesterone/ml (P<0.01) were secreted. Luteal cells from the 13th day of the estrous cycle incubated without prolactin secreted on average 70.72 ± 9.21 ng progesterone/ml of medium, whereas under the influence of different prolactin doses 50.75 ± 8.52, 46.54 ± 7.13, 43.30 ± 6.78 and 41.68 ± 7.21 ng progesterone/ml (P<0.01) were secreted.Prolactin did not change progesterone secretion by luteal cells obtained on the 17th day of the estrous cycle. An influence of the incubation time on progesterone secretion by these cells was observed: after 30 min of incubation the cells secreted 8.83 ± 2.95 ng/ml, after 3 h 8.12 ± 2.57 ng/ml and after 6 h 6.86 ± 1.91 ng/ml, irrespective of the amount of PRL added.The results suggest that prolactin plays a role in the luteolysis of the corpus luteum.     

Osmolyte channel regulation by ionic strength in skate RBC

The aim of this study was to determine whether the opening of the osmolyte channel in skate red blood cells (RBC) is regulated by intracellular electrolyte concentration and conductivity. Consistent with previous studies, experiments with hyperosmotic preincubation before cell swelling or swelling with an isosmotic electrolyte (e.g., ammonium chloride) showed that an increase in ionic strength inhibits the opening of the taurine channel. However, a decrease in intracellular ionic strength did not always stimulate taurine efflux to the same degree. Whereas hyposmotic swelling caused a large increase in taurine efflux, swelling induced by treatment with isosmotic nonelectrolytes produced much smaller stimulation. Results with assays for band 3 phosphorylating enzymes were consistent with those from the taurine efflux studies; stimulation of enzyme activity was lower in cells that were swollen with isosmotic nonelectrolyte media than in cells swollen in hyposmotic media. These results indicate that a decrease in ionic strength is not the only signal for the opening of the taurine channel in skate RBC. Ionic strength does affect channel activity, but there must also be some other regulator.     

A novel running wheel apparatus to monitor locomotor rhythms in land crabs

The spatial distribution of cephalopods is influenced by salinity and temperature. In marine osmoconformers, one reason for different spatial distributions maybe the putative distinct capacity to regulate tissue hydration. Adult Octopus vulgaris (n?=?6) and O. insularis (n?=?6) were obtained by divers from sites along the southern and northeastern Brazilian coasts, respectively. In the laboratory, octopuses were acclimated to full-strength seawater (salinity 34), for 5–10 days. They were then anesthetized (10 min in 4 °C seawater), a portion of the second right arm was removed, and cut into three portions. Tissues were placed in control isosmotic saline (osmolality 1072 mOsm/kg H₂O). Fragments were weighed and transferred to either isosmotic, hyposmotic (50%), or hyperosmotic (150%) salines, and their weight was observed for 120 min. Tissue from both species maintained their hydration/weight following the hyposmotic shock, but differed in their response to the hyperosmotic challenge, where hydration remained unchanged in O. insularis, but was significantly reduced in O. vulgaris. This result could partially explain why O. insularis is more abundant than O. vulgaris throughout the shallow reefs and tide pools in the warmer and salty tropical waters of the northeastern Brazilian coast.     

The effect of hyposmotic and isosmotic cell swelling on the intracellular [Ca2+] in lactating rat mammary acinar cells

The effect of hyposmotic and isosmotic cell swelling on the free intracellular calcium concentration ([Ca²⁺]_i) in rat mammary acinar cells has been examined using the fura-2 dye technique. A hyposmotic shock (40% reduction) increased the [Ca²⁺]_i in rat mammary acinar cells in a fashion which was transient; the [Ca²⁺]_i returned to a value similar to that found under isomotic conditions within 180 sec. The increase in the [Ca²⁺]_i was dependent upon the extent of the osmotic shock. The hyposmotically-activated increase in the [Ca²⁺]_i could not be attributed to a reduction in extracellular Na⁺ or a change in the ionic strength of the incubation medium. Thapsigargin (1 M) enhanced the hyposmotically-activated increase in the [Ca²⁺]_i. Isosmotic swelling of rat mammary acinar cells, using urea, had no significant effect on the [Ca²⁺]_i. Similarly, a hyperosmotic shock did not affect the [Ca²⁺]_i in rat mammary acinar cells. It appears that the effect of cell swelling on the [Ca²⁺]_i in rat mammary acinar cells depends on how the cells are swollen (hyposmotic vs. isosmotic). This finding may have important physiological implications given that it is predicted that mammary cell volume will change in vivo under isomotic conditions.