Isosmotic Conditions Research Articles

Influence of salinity and cadmium on the survival and osmoregulation of Callianassa kraussi and Chiromantes eulimene (Crustacea: Decapoda)

This study investigated the influence of salinity and cadmium on the survival and osmoregulatory capability of two decapod crustaceans, Callianassa kraussi and Chiromantes eulimene. Callianassa kraussi was able to survive in salinities of 5–55 over 96 h, whilst C. eulimene survived in 0–55 over the same time period. The 96-hour cadmium LC50 for both species decreased progressively at salinities above and below their respective isosmotic conditions, with the decrease being slightly more pronounced below compared to above isosmotic salinity. A hypo-iso-osmoregulatory strategy was followed by C. kraussi as it hyper-osmoregulated at salinities between 5 and 25 and osmoconformed at salinities greater than 25. Chiromantes eulimene followed a hyper-hypo-osmoregulatory strategy; it hyper-regulated in salinities from 0 up to isosmotic conditions at about 28 (c. 780 mOsm kg−1), followed by hypo-regulation up to 55. The effect of cadmium exposure on the osmoregulatory capacity of C. kraussi was more pronounced at hyper-regulating salinities (5–25) whilst on C. eulimene the influence was more pronounced at salinities above the isosmotic point (28). The influence of salinity and cadmium on both survival and osmoregulation of the two crustaceans are discussed by outlining the chemical and physiological mechanisms involved.

Potentiation of Regulatory Volume Decrease by a P2-Like Receptor and Arachidonic Acid in American Alligator Erythrocytes

This study examined the role of a P2 receptor and arachidonic acid (AA) in regulatory volume decrease (RVD) by American alligator red blood cells (RBCs). Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca(2+) concentration were visualized using fluorescence microscopy. Gadolinium (50μM), hexokinase (2.5U/ml), and suramin (100μM) increased osmotic fragility, blocked volume recovery after hypotonic shock, and prevented a rise in intracellular Ca(2+) that normally occurs during cell swelling. The P2X antagonists PPADS (50μM) and TNP-ATP (10μM) also increased fragility and inhibited volume recovery. In contrast, ATPγS (10μM), α,β-methylene-ATP (50μM) and Bz-ATP (50μM) had the opposite effect, whereas 2-methylthio-ATP (50μM) and UTP (10μM) had no effect. In addition, the phospholipase A(2) (PLA(2)) inhibitors ONO-RS-082 (10μM), chlorpromazine (10μM), and isotetrandrine (10μM) increased osmotic fragility and blocked volume recovery, whereas AA (10μM) and its nonhydrolyzable analog eicosatetraynoic acid (ETYA, 10μM) had the reverse effect. Further, AA (10μM), but not ATPγS (10μM), prevented the inhibitory effect of a low Ca(2+)-EGTA Ringer on RVD, whereas both AA (10μM) and ATPγS (10μM) caused cell shrinkage under isosmotic conditions. In conclusion, our results are consistent with the presence of a P2-like receptor whose activation stimulated RVD. In addition, AA also was important for volume recovery.

Optimal concentrations of cryoprotective agents for semen from stallions that are classified ‘good’ or ‘poor’ for freezing

Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as ‘poor’ freezers, and when post-thaw viability is high they are designated as ‘good’ freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is exposed to during cryopreservation, and to determine if sperm from ‘good’ and ‘poor’ freezers show differences in osmotic tolerance limits and in the suitability of cryoprotective agents. Concentrations of 2–3% of the above mentioned cryoprotectants with freezing extender osmolalities ranging from 580 to 895 mOsm kg −1 showed the highest motility rates after freeze–thaw, both for ‘good’ and ‘poor’ freezers, for all cryoprotectants tested with slightly higher values for glycerol. Freeze–thawed semen from ‘poor’ freezers was found to have a lower percentage of progressively motile sperm compared to that of ‘good’ freezers. Assessment of plasma and acrosomal membrane integrity after return to isosmotic conditions revealed that cryopreserved sperm from ‘poor’ freezers showed lower osmotic tolerance limits as compared to sperm from ‘good’ freezers. Semen from ‘poor’ freezers that was frozen using freezing extenders supplemented with more then 2% cryoprotectant showed decreased viability and increased acrosome reaction upon return to isoosmotic conditions, whereas ‘good’ freezers could withstand cryoprotectant concentrations up to 3% before a decline in viability was observed.

Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+exchange

Na(+)-K(+)-2Cl(-) cotransport (NKCC) mediates the movement of two Cl(-) ions for one Na(+) and one K(+) ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl(-) uptake in Xenopus laevis oocytes, as measured using (36)Cl, is twice the value of K(+) uptake, as determined using (86)Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K(+) uptake with only a minimal increase in bumetanide-sensitive Cl(-) uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K(+)/K(+) and Cl(-)/Cl(-) exchange. Although significant stimulation of K(+)/K(+) exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K(+)/K(+) exchange.

HIV protease inhibitors elicit volume-sensitive Cl− current in cardiac myocytes via mitochondrial ROS

HIV protease inhibitors (HIV PI) reduce morbidity and mortality of HIV infection but cause multiple untoward effects. Because certain HIV PI evoke production of reactive oxygen species (ROS) and volume-sensitive Cl− current (ICl,swell) is activated by ROS, we tested whether HIV PI stimulate ICl,swell in ventricular myocytes. Ritonavir and lopinavir elicited outwardly rectifying Cl− currents under isosmotic conditions that were abolished by the selective ICl,swell-blocker DCPIB. In contrast, amprenavir, nelfinavir, and raltegravir, an integrase inhibitor, did not modulate ICl,swell acutely. Ritonavir also reduced action potential duration, but amprenavir did not. ICl,swell activation was attributed to ROS because ebselen, an H2O2 scavenger, suppressed ritonavir- and lopinavir-induced ICl,swell. Major ROS sources in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin failed to block ritonavir- or lopinavir-induced currents, although it blocks ICl,swell elicited by osmotic swelling or stretch. In contrast, rotenone, a mitochondrial e− transport inhibitor, suppressed both ritonavir- and lopinavir-induced ICl,swell. ROS production was measured in HL-1 cardiomyocytes with C-H2DCFDA-AM and mitochondrial membrane potential (ΔΨm) with JC-1. Flow cytometry confirmed that ritonavir and lopinavir but not amprenavir, nelfinavir, or raltegravir augmented ROS production, and HIV PI-induced ROS production was suppressed by rotenone but not NADPH oxidase blockade. Moreover, ritonavir, but not amprenavir, depolarized ΔΨm. These data suggest ritonavir and lopinavir activated ICl,swell via mitochondrial ROS production that was independent of NADPH oxidase. ROS-dependent modulation of ICl,swell and other ion channels by HIV PI may contribute to some of their actions in heart and perhaps other tissues.

Endothelin signalling regulates volume-sensitive Cl− current via NADPH oxidase and mitochondrial reactive oxygen species

We assessed regulation of volume-sensitive Cl(-) current (I(Cl,swell)) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes. ET-1 elicited I(Cl,swell) under isosmotic conditions. Outwardly rectified Cl(-) current was blocked by the I(Cl,swell)-selective inhibitor DCPIB or osmotic shrinkage and involved ET(A) but not ET(B) receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of I(Cl,swell) by osmotic swelling or angiotensin II (AngII) was suppressed by ET(A) blockade, whereas AngII AT(1) receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I(Cl,swell). As expected, blockade of NOX suppressed ET-1-induced I(Cl,swell), but blockade of mitochondrial ROS production with rotenone also suppressed I(Cl,swell). I(Cl,swell) was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I(Cl,swell) was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade. ET-1-ET(A) signalling activated I(Cl,swell) via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ET(A) receptors were downstream effectors when I(Cl,swell) was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I(Cl,swell) is likely to participate in multiple physiological and pathophysiological processes.

MOLECULAR DETERMINANT(S) OF HYPER‐OSMOTICALLY ACTIVATED NKCC1‐MEDIATED K+/K+ EXCHANGE

Na+‐K+‐2Cl− cotransport mediates the movement of two Cl− ions for one Na+ and one K+ ion. Under isosmotic conditions or SPAK/WNK4 activation, the NKCC1‐mediated bumetanide‐sensitive Cl− uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. In contrast, under hyperosmotic conditions, there is a very significant activation of the bumetanide‐sensitive K+ uptake with only a minimal increase in Cl− uptake. This indicates that when stimulated by hypertonicity, the cotransporter mediates Na+‐K+‐2Cl− cotransport as well as K+/K+ exchange. Interestingly, this stimulation of K+/K+ exchange was only observed with NKCC1, but not NKCC2. In order to identify the molecular determinant(s) of this NKCC1‐specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2, nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the second extracellular loop between membrane spanning domains three and four is involved in the hyperosmotic stimulation of K+ uptake (K+/K+ exchange) in NKCC1. However, replacing only this loop in NKCC2 was insufficient for K+ uptake activation, suggesting an interaction between this loop and a yet to be identified region of the cotransporter.

Principles and perspectives for the conservation of goat buck spermatozoa

This paper is focused on the use of alternative protocols for goat buck sperm cryopreservation and other current approaches to study sperm cryosurvival, considering theoretical principles for the preservation of cells at freezing temperatures. Simple modifications to traditional freeze-thawing protocols may improve sperm cryosurvival; thus, cooling to −5 °C instead of +5 °C, before freezing, improves buck, ram and boar sperm cryosurvival. Premature capacitation induced by cryopreservation may be used as an additional tool to: (1) assess new cryopreservation protocols; i.e. the least aggressive one would induce the lowest proportion of premature capacitated spermatozoa; (2) to estimate the subpopulation of frozen-thawed spermatozoa that remain fertile (live cells showing Pattern F or B; Chlortetracycline Assay). Freezing and storage of spermatozoa using ultra-low freezers (−150 °C) represents a feasible alternative to the use of liquid nitrogen, at least in both short and medium term. Simple tests based on simulation of osmotic stress that occurs during freeze-thawing are promising approaches to predict sperm cryosurvival. Assessment of spermatozoa during hyperosmotic and isosmotic conditions at 23 °C has revealed that proportions of plasma membrane-intact and acrosome-intact spermatozoa are similar to those observed after cryopreservation (after freeze-thawing). Inter-male differences in sperm freezability are an ever present variable influencing sperm cryosurvival.

Sphingosine-1-Phosphate Regulates Volume-Sensitive Chloride Current in Ventricular Myocytes by Means of ROS

We previously demonstrated that bacterial sphingomyelinase (SMase) activates a volume-sensitive current, ICl,swell, by a pathway that involves mitochondrial ROS production. SMase activity generates endogenous ceramides from sphingomyelin in the outer plasma membrane leaflet and, in turn, ceramides are metabolized to several sphingolipids, including sphingosine-1-phosphate (S1P). We tested whether ceramide metabolites are responsible for eliciting ICl,swell. Under isosmotic conditions that isolate anion currents, SMase-induced ICl,swell was abrogated by blockade of ceramidase (converts ceramide to sphingosine) with D-erythro-MAPP (10 μM). SMase-induced ICl,swell also was suppressed by inhibition of sphingosine kinase with DL-threo-dihydrosphingosine (10 μM). These data suggested that the ceramide metabolite S1P is likely to stimulate ICl,swell. As expected, exogenous S1P (500 nM) elicited an outwardly rectifying Cl- current that was fully inhibited by the ICl,swell-specific blocker DCPIB (10 μM). As seen with SMase-induced ICl,swell, S1P-induced ICl,swell was fully inhibited by the mitochondrial Complex I blocker rotenone (10 μM), which suppresses extramitochondrial ROS release by Complex III. In contrast to results with SMase, S1P-induced current was partially inhibited by blockade of NADPH oxidase (NOX) with apocynin (500 μM). These data indicate that S1P is a necessary component of SMase-induced ICl,swell activation and that the action of exogenous S1P involves ROS from both mitochondria and NOX. Importantly, exogenous C2-ceramide (2 μM), a synthetic short-chain ceramide, also elicits ICl,swell even though C2-ceramide is not metabolized to S1P in native cells. Thus, it seems likely that ceramides can elicit ICl,swell via S1P and also by a distinct pathway and that both pathways converge at mitochondrial ROS.

Regulation of paracellular Na+ and Cl− conductances by hydrostatic pressure

The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na(+) conductance (G(Na)) and the Cl(-) conductance (G(Cl)) has been Investigated. Gp, G(Na) and G(Cl) were time-dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1-4cm H2O) applied from the basolateral side enhanced the osmotic gradient-induced increase in Gp, G(Na) and G(Cl) in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, G(Na) and G(Cl). How the hydrostatic pressure influences Gp, G(Na) and G(Cl) under an isosmotic condition was also investigated. Gp, G(Na) and G(Cl) were stably constant under a condition with basolateral application of sucrose canceling the NaCl-generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, G(Na) and G(Cl), while apically applied hydrostatic pressure had little effect on Gp, G(Na) or G(Cl). Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, G(Na) and G(Cl).

Whole-Cell Patch Clamping of Isolated Fiber Cells Confirms that Spatially Distinct Cl−Influx and Efflux Pathways Exist in the Cortex of the Rat Lens

To test the hypothesis that lens fiber cells use different combinations of transport proteins to mediate Cl influx and efflux in order to regulate their steady state volume. Cells were isolated from rat lenses by enzymatic dissociation in the presence of Gd(3+), and short and long fiber cells were assigned to peripheral efflux and deeper influx zones, respectively. Electrical properties were of isolated cells, and whole lenses were analyzed by using whole-cell patch clamping and intracellular microelectrodes, respectively, before and after exposure to hyposmotic challenge and/or the addition of [(dihydronindenyl)oxy] alkanoic acid (DIOA). Cells from the influx zone were dominated by an outwardly rectifying Cl(-) conductance, and exposure to hyposmotic challenge increased this conductance. Cells isolated from the efflux zone were dominated by K(+) conductance(s) with only a minimal contribution from the Cl(-) conductance. Exposure of cells that exhibited a minimal baseline Cl(-) conductance to hyposmotic challenge caused swelling and a transient increase in Cl(-) current. In other cells that initially lacked a Cl(-) conductance, hyposmotic challenge caused swelling, but no increase in outward current. However, the subsequent addition of DIOA exacerbated swelling and activated a Cl(-) current. Under isosmotic conditions, addition of DIOA also induced cell swelling and the transient activation of a Cl(-) current. In whole lenses, exposure to hyposmotic challenge increased the contribution of an anion conductance to the membrane potential. In peripheral cells, Cl(-) efflux is primarily mediated by potassium chloride cotransporters (KCCs) and its activity can be upregulated by hyposmotic challenge. In addition, these cells also contain a Cl(-) channel that exhibits a variable baseline activity level and that can be recruited to effect regulatory volume decrease if the KCC transporters are inhibited.

Endothelin-1 Regulates Volume-Sensitive Chloride Current in Rabbit Atrial Myocytes via Reactive Oxygen Species from Mitochondria and NADPH Oxidase

Angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are implicated in the activation of volume-sensitive Cl current (ICl,swell) by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ET-1 blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell. Under isosmotic conditions, ET-1 (10 nM) elicited an outwardly rectifying Cl current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB (10 μM) and by osmotic shrinkage. Selective ETA (BQ-123, 1 μM) but not ETB blockade (BQ-788, 100 nM) fully suppressed ET-1-induced current. ET-1-induced ICl,swell also was abolished by inhibitors of EGFR kinase (AG1478, 10 μM) and PI-3K (LY294002, 20 μM; wortmannin, 500 nM), which also suppress stretch- and swelling-induced ICl,swell. ERK inhibitors (PD 98059, 10 μM; U0216, 1 μM) partially and fully blocked ET-1- and EGF- induced currents, respectively, but did not effect ICl,swell elicited by H2O2. ET-1 acts downstream from AngII. ETA blockade (BQ-123) abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade (losartan, 5 μM) did not effect ET-1-induced ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin (500 μM) or mitochondrial complex I with rotenone (10 μM) both completely suppressed ET-1-induced ICl,swell. In contrast, ICl,swell elicited by antimycin A (10 μM), which stimulates superoxide production by mitochondrial complex III, was insensitive to apocynin and the NOX fusion peptide inhibitor gp91ds-tat (500 nM). These data suggest that ET-1 and ETA receptors are required intermediates in AngII-, swelling-, and stretch-induced activation of ICl,swell. Moreover, enhancement of mitochondrial ROS production by ROS from NOX is likely to contribute to activation of ICl,swell by ET-1.

Ceramide Activates ICl,swell in Rabbit Ventricular Myocytes via Mitochondrial ROS Production

We previously showed that ICl,swell is activated by ROS generation via NADPH oxidase (NOX) and the mitochondrial electron transport chain (ETC). Sphingolipid signaling is implicated in channel regulation. Here we examined the role of ceramide in the modulation of ICl,swell. Under isosmotic conditions, the addition of exogenous C2-ceramide (C2-Cer, 2 ∝M) increased Cl− current density by 0.7 ± 0.1 pA/pF at +60 mV after 10 min (n = 11, P<0.01). DCPIB (10 ∝M), a highly selective ICl,swell antagonist, inhibited C2-Cer-induced ICl,swell by 76 ± 8% (n = 6, P<0.01). The inactive analogue C2-H2Cer (2 ∝M) failed to stimulate ICl,swell (n = 6). Bacterial sphingomyelinase (SMase, 0.03 U/mL) was used to elicit endogenous ceramide production. SMase increased ICl,swell by 1.1 ± 0.1 pA/pF after 14 min (n = 30, P<0.01). This activation was inhibited by DCPIB (78 ± 6%, 10 ∝M, n = 7 and 81 ± 6%, 30 ∝M, n = 4) and tamoxifen (116 ± 16%, 10 ∝M, n = 5). Next we identified the source of ROS. Exposure to the NOX-specific inhibitor apocynin (500 ∝M, 10 min) failed to suppress SMase-induced ICl,swell (n = 9), whereas we previously showed apocynin blocks activation of ICl, swell on swelling and stretch. Diphenyleneiodonium (60 ∝M), a flavoprotein oxidase antagonist that suppresses both NOX and ETC Complex I, fully inhibited SMase-induced ICl,swell after 20 min (100 ± 14%, n = 4, P<0.01). Rotenone (10 ∝M), a specific ETC Complex I inhibitor, also abrogated the SMase-induced activation of ICl,swell after 20 min (110 ± 18%, n = 5, P<0.05). These data indicate that there is a ceramide-sensitive component of ICl,swell, and it is regulated by mitochondrial ROS. Ceramide signaling may modulate ICl,swell in cardiac disease.

HIV Protease Inhibitors Activate Volume-Sensitive Chloride Current in Ventricular Myocytes by Generating Mitochondrial Reactive Oxygen Species

HIV protease inhibitors (HIV PI) have been successfully used to reduce morbidity and mortality of HIV infection. However, their long-term use causes significant side effects including cardiac arrhythmias. Previously we showed that outwardly-rectifying, volume-sensitive Cl current (ICl,swell) is regulated by signaling pathways that elicit production of reactive oxygen species (ROS). Because certain HIV PI recently were reported to augment ROS production, we tested whether HIV PI stimulate ICl,swell in rabbit ventricular myocytes. Under isosmotic conditions, ritonavir (15 μM, 20 min) and lopinavir (15 μM, 25 min) induced outwardly-rectifying Cl currents (1.5 ± 0.3 pA/pF and 1.9 ± 0.3 pA/pF at +60 mV, respectively) that were fully inhibited by the highly selective ICl,swell-blocker DCPIB (10 μM). In contrast, amprenavir (15 μM, 30 min) and nelfinavir (15 μM, 30 min) did not modulate ICl,swell, and raltegravir (MK-0518, 15 μM, 30 min), an HIV integrase inhibitor, also was ineffective. Two major sources of ROS in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin (500 μM) failed to inhibit the ritonavir- or lopinavir-induced currents, although we previously found apocynin blocks ICl,swell activation upon osmotic swelling and stretch. In contrast, rotenone (10 μM, 30 min), a mitochondrial complex I inhibitor that limits electron flux to and ROS production by complex III, blocked 102 ± 4% of ritonavir- and 82 ± 12% of lopinavir-induced ICl,swell. Furthermore, the membrane-permeant, glutathione peroxidase mimetic ebselen (15 μM, 15 min) suppressed ICl,swell elicited by ritonavir (102 ± 3%) and lopinavir (93 ± 6%). These results suggest that ritonavir and lopinavir activate ICl,swell via mitochondrial ROS production by complex III. Activation of ICl,swell by certain HIV PI may contribute to their untoward effects in heart and potentially other tissues.

80 CHOLESTEROL-LOADED METHYL-BETA-CYCLODEXTRIN IMPROVES BOAR SPERMATOZOA CRYOPROTECTANT ADDITION AND REMOVAL TOLERANCE

Difficulties associated with the cryopreservation of boar sperm include their sensitivities to osmotic stresses and chilling sensitivity. We investigated the effects of cholesterol-loaded cyclodextrin (CLC) on boar sperm motility and membrane integrity following exposure to various osmolalities. Samples were collected using the gloved hand method from crossbred boars, and ejaculates having greater than 75% motility were extended 1:3 with Androhep (Minitube Inc., Verona, WI) for this study. Samples were centrifuged at 700g for 5 min, and the resulting pellets were resuspended to 1.2 × 108 cells mL–1 in Androhep. Samples were then treated with 0, 1.5, or 3.0 mg of CLC/1.2 × 108 cells mL–1 for 10 min at room temperature. In experiment 1, samples were aliquoted into 1.5-mL centrifuge tubes, centrifuged at 700g for 5 min and the sperm exposed to Dulbecco’s PBS at different osmolalities for 5 min before being returned to 300 mOsm by adding Dulbecco’s PBS solutions at differing osmolalities. After returning the sperm to isosmotic conditions, sperm motility was analyzed. In experiment 2, samples were treated as in experiment 1 and following exposure to the various osmolalities, sperm were stained with Alexa 488-PNA and propidium iodide to determine sperm membrane integrity. Ten thousand sperm per treatment were analyzed by flow cytometry. Data were analyzed by standard ANOVA. The CLC-treated sperm (normalized means ± SEM; 33 ± 16, 80 ± 8, 86 ± 5, 100, 64 ± 4, 7 ± 3, 0 ± 0, respectively) exhibited greater percentages of motile cells following hypo-isosmotic exposure than control sperm (4 ± 1.6, 33 ± 9.6, 84 ± 7.1, 100, 37 ± 5.5, 3 ± 1.6, 0 ± 0, respectively), and there was a tendency for CLC-treated sperm (P = 0.0225) to maintain motility following hyper-isosmotic exposure. In addition, CLC-treated sperm (87 ± 4, 93 ± 1, 95 ± 1, 93 ± 2, 88 ± 4, 83 ± 3, 41 ± 9, respectively; P &lt; 0.05) maintained greater percentages of membrane integrity following treatment with anisosmotic solutions compared with controls (29 ± 8, 63 ± 10, 81 ± 7, 92 ± 3, 73 ± 8, 44 ± 5, 21 ± 9, respectively). Using a combination of these osmotic tolerance data with previously published boar sperm membrane permeability characteristics, we mathematically modeled the number of steps needed for the addition or removal of cryoprotectants. Computer simulations indicate that an abrupt addition of 1 m glycerol will cause boar sperm to exceed their osmotic tolerance limits unless they are treated with 3 mg of CLC. Moreover, the addition of 1 m EG causes boar sperm to exceed all osmotic tolerance limits and therefore, the addition and removal of EG requires multiple-step protocols. However, the addition and removal of 1 m DMSO maintains volume excursions well within the osmotic tolerance limits with the addition of cholesterol (1.5 and 3 mg). Empirical data for addition of CPA have shown similar results as seen with the computer simulation. These data support the hypothesis that adding cholesterol to porcine sperm broadens their osmotic tolerance limits and potentially provide a mechanism to increase post-thaw survival of porcine sperm.

Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures

Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following artificial insemination (AI), are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPAs) and chilling on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In experiment 1, ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900 and 1200 milliosmolal (mOsm)/kg sucrose solutions, held for 5 min and then returned to isosmotic condition. Motility characteristics of sperm during exposure to each anisosmotic solutions and after returning to isosmotic conditions were determined. In experiment 2, ejaculated and epididymal ram sperm were exposed to 1 M glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after returning to isosmotic conditions were determined. In experiment 3, effects of various temperatures on motility characteristics of ejaculated and epididymal ram sperm were determined after exposing them to three different sub-physiologic temperatures (4, 10 and 22 °C) for 30 min and subsequently returning them to 37 °C. The motility of ejaculated ram sperm was significantly more affected from anisosmotic stress than was epididymal ram sperm ( P < 0.05). While anisosmotic stress had no effects on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1 M Gly, DMSO, EG or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm ( P > 0.05). However, there was a slight decrease in acrosomal integrity for ejaculated ram sperm after exposure to 1 M Gly, DMSO or EG ( P > 0.05). Both epididymal and ejaculated ram sperm exhibited temperature-dependent loss of motility and acrosomal integrity ( P < 0.05). However, ejaculated ram sperm was more sensitive to chilling stress than epididymal sperm ( P < 0.05). In conclusion, the current data suggest that while epididymal ram sperm is extremely resilient to various cryobiologically relevant stress conditions, ejaculated ram sperm demonstrate greater sensitivity to such stressors. These findings should be taken into account when developing cryopreservation protocols for ejaculated and epididymal ram sperm.

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The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

Regulatory volume decrease after swelling induced by urea in fibroblasts: prominent role of organic osmolytes

Cell swelling, regulatory volume decrease (RVD), volume-sensitive Cl(-) (Cl(-) (swell)) current and taurine efflux after exposure to high concentrations of urea were characterized in fibroblasts Swiss 3T3, and results compared to those elicited by hyposmotic (30%) swelling. Urea 70, 100, and 150 mM linearly increased cell volume (8.25%, 10.6%, and 15.7%), by a phloretin-inhibitable process. This was followed by RVD by which cells exposed to 70, 100, or 150 mM urea recovered 27.6%, 38.95, and 74.1% of their original volume, respectively. Hyposmolarity (30%) led to a volume increase of 25.9% and recovered volume in 32.5%. (3)H-taurine efflux was increased by urea with a sigmoid pattern, as 9.5%, 18.9%, 71.5%, and 89% of the labeled taurine pool was released by 70, 100, 150, or 200 mM urea, respectively. Only about 11% of taurine was released by 30% hyposmolarity reduction in spite of the high increase in cell volume. Urea-induced taurine efflux was suppressed by NPPB (100 microM) and markedly reduced by the tyrosine kinase-general blocker AG18. The Cl(-) (swell) current was more rapidly activated and higher in amplitude in the hyposmotic than in the isosmotic/urea condition (urea 150 mM), but this was not sufficient to accomplish an efficient RVD. These results showed that at similar volume increase, cells swollen by urea showed higher taurine efflux, lower Cl(-) (swell) current and more efficient RVD, than in those swollen by hyposmolarity. The correlation found between RVD efficiency and taurine efflux suggest a prominent role for organic over ionic osmolytes for RVD evoked by urea in isosmotic conditions.

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 Ba(2+), 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 NH(2)-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.

Thrombin potently enhances swelling‐sensitive glutamate efflux from cultured astrocytes

High concentrations of thrombin (Thr) have been linked to neuronal damage in cerebral ischemia and traumatic brain injury. In the present study we found that Thr markedly enhanced swelling-activated efflux of (3)H-glutamate from cultured astrocytes exposed to hyposmotic medium. Thr (0.5-5 U/mL) elicited small (3)H-glutamate efflux under isosmotic conditions and increased the hyposmotic glutamate efflux by 5- to 10-fold, the maximum effect being observed at 15% osmolarity reduction. These Thr effects involve its protease activity and are fully mimicked by SFFLRN, the synthetic peptide activating protease-activated receptor-1. Thr potentiation of (3)H-glutamate efflux was largely dependent on a Thr-elicited increases in cytosolic Ca(2+) (Ca(2+) (i)) concentration ([Ca(2+)](i)). Preventing Ca(2+) (i) rise by treatment with EGTA-AM or with the phospholipase C blocker U73122 reduced the Thr-increased glutamate efflux by 68%. The protein kinase C blockers Go6976 or chelerythrine reduced the Thr effect by 19%-22%, while Ca/calmodulin blocker W7 caused a 63% inhibition. In addition to this Ca(2+)-sensitive pathway, Thr effect on glutamate efflux also involved activation of phosphoinositide-3 kinase (PI3K), since it was reduced by the PI3K inhibitor wortmannin (51% inhibition). Treating cells with EGTA-AM plus wortmannin essentially abolished Thr-dependent glutamate efflux. Thr-activated glutamate release was potently inhibited by the blockers of the volume-sensitive anion permeability pathway, NPPB (IC(50) 15.8 microM), DCPIB (IC(50) 4.2 microM), and tamoxifen (IC(50) 6.6 microM. These results suggest that Thr may contribute to the excitotoxic neuronal injury by elevating extracellular glutamate release from glial cells. Therefore, this work may aid in search of neuroprotective strategies for treating cerebral ischemia and brain trauma.