Lipids of the liver microsomal fraction in the ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis> during hibernation

Winter sleep of the ground squirrel Spermophilus undulatus was accompanied by a 20% decrease in phospholipid content (µg phospholipid per 1 mg protein) in microsomal fractions of the liver as compared with summer-active squirrels. The phosphatidylcholine level (mol %) in hibernating squirrels was lower than in summer-active squirrels, and the content of sphingomyelin (mol %) during the torpor bout was higher than in winter- and summer-active squirrels. The cholesterol, fatty acid, monoglyceride, and diglyceride levels in the microsomal fraction of the liver were elevated during hibernation. Pronounced seasonal changes in the lipid/protein ratio implicate the lipids of the liver microsomal fraction in adaptation of the ground squirrel to hibernation.     

Hemoglobin alterations of the 13-lined ground squirrel while in various activity states.

1. Characterization of fetal, winter-hibernating, winter-active, summer-active and summer-induced hibernating hemoglobins of 13-lined ground squirrels (Citellus tridecemlineatus) by isoelectric focusing (IEF) pH 7.0-9.0 indicated that this molecule is extremely responsive to the various activity states of this hibernator. 2. Major alterations of ground squirrel hemoglobin occur with the varying activity states as evidenced by the distinctive changes in the isoelectric points (pIs) of these protein components. 3. Hemoglobin from winter-hibernating or summer-induced hibernating ground squirrels does not revert to a fetal type of hemoglobin. 4. The presence of an additional hemoglobin peak pI 6.55 in the summer-induced hibernator may serve as a possible assay for hibernation inducing trigger(s) (HIT) molecules under study in our laboratory.     

Adaptation to low body temperature influences pulmonary surfactant composition thereby increasing fluidity while maintaining appropriately ordered membrane structure and surface activity

The interfacial surface tension of the lung is regulated by phospholipid-rich pulmonary surfactant films. Small changes in temperature affect surfactant structure and function in vitro. We compared the compositional, thermodynamic and functional properties of surfactant from hibernating and summer-active 13-lined ground squirrels (Ictidomys tridecemlineatus) with porcine surfactant to understand structure-function relationships in surfactant membranes and films. Hibernating squirrels had more surfactant large aggregates with more fluid monounsaturated molecular species than summer-active animals. The latter had more unsaturated species than porcine surfactant. Cold-adapted surfactant membranes displayed gel-to-fluid transitions at lower phase transition temperatures with reduced enthalpy. Both hibernating and summer-active squirrel surfactants exhibited lower enthalpy than porcine surfactant. LAURDAN fluorescence and DPH anisotropy revealed that surfactant bilayers from both groups of squirrels possessed similar ordered phase characteristics at low temperatures. While ground squirrel surfactants functioned well during dynamic cycling at 3, 25, and 37°C, porcine surfactant demonstrated poorer activity at 3°C but was superior at 37°C. Consequently the surfactant composition of ground squirrels confers a greater thermal flexibility relative to homeothermic mammals, while retaining tight lipid packing at low body temperatures. This may represent the most critical feature contributing to sustained stability of the respiratory interface at low lung volumes. Thus, while less effective than porcine surfactant at 37°C, summer-active surfactant functions adequately at both 37°C and 3°C allowing these animals to enter hibernation. Here further compositional alterations occur which improve function at low temperatures by maintaining adequate stability at low lung volumes and when temperature increases during arousal from hibernation.     

Increased Na+/Ca2+ Exchanger Activity Promotes Resistance to Excitotoxicity in Cortical Neurons of the Ground Squirrel (a Hibernator)

Ground squirrel, a hibernating mammalian species, is more resistant to ischemic brain stress than rat. Gaining insight into the adaptive mechanisms of ground squirrels may help us design treatment strategies to reduce brain damage in patients suffering ischemic stroke. To understand the anti-stress mechanisms in ground squirrel neurons, we studied glutamate toxicity in primary cultured neurons of the Daurian ground squirrel (Spermophilus dauricus). At the neuronal level, for the first time, we found that ground squirrel was more resistant to glutamate excitotoxicity than rat. Mechanistically, ground squirrel neurons displayed a similar calcium influx to the rat neurons in response to glutamate or N-methyl-D-aspartate (NMDA) perfusion. However, the rate of calcium removal in ground squirrel neurons was markedly faster than in rat neurons. This allows ground squirrel neurons to maintain lower level of intracellular calcium concentration ([Ca²⁺]_i) upon glutamate insult. Moreover, we found that Na⁺/Ca²⁺ exchanger (NCX) activity was higher in ground squirrel neurons than in rat neurons. We also proved that overexpression of ground squirrel NCX2, rather than NCX1 or NCX3, in rat neurons promoted neuron survival against glutamate toxicity. Taken together, our results indicate that ground squirrel neurons are better at maintaining calcium homeostasis than rat neurons and this is likely achieved through the activity of ground squirrel NCX2. Our findings not only reveal an adaptive mechanism of mammalian hibernators at the cellular level, but also suggest that NCX2 of ground squirrel may have therapeutic value for suppressing brain ischemic damage.     

Seasonal activity patterns of glycolytic enzymes in a hibernator,the Arctic ground squirrel Spermophilus parryi

Summary The maximum activities of the enzymes of glycolysis of five tissues (cerebrum, heart, liver, kidney cortex and skeletal muscle) were examined in a hibernator, the Arctic ground squirrel, in both the summer-active and hibernating (winter) states. In addition, by observing through electrofocussing the appearance of variants of hepatic pyruvate kinase, an attempt was made to determine the time-course of preparation for hibernation. This process requires about 4 weeks, which is about the same as that for the preparation for emergence from hibernation. The separate tissues responded in individually characteristic fashions, although the enzymes from kidney cortex and liver tended to show a general increase and the enzymes from heart and brain showed a general decrease. The enzyme activities in skeletal muscle changed in seemingly disparate ways with some enzymes increasing in hibernation, while others decreased, and yet others remained unchanged. These results are discussed in the light of the hibernating habit of the mammal.     

Mitochondrial metabolism in hibernation: metabolic suppression, temperature effects, and substrate preferences

We compared liver and skeletal muscle mitochondrial function among activity states to characterize regulated reversible metabolic suppression in the mammalian hibernator Spermophilus tridecemlineatus. At 37 degrees C, succinate oxidation was 70% lower in the liver mitochondria from torpid animals than in those from summer-active animals or in animals arousing from torpor. Respiration was very sensitive to temperature (Q(10) 5.8-9.8), and when measured at 25 degrees or 5 degrees C there was no difference among the three states. Liver mitochondria from summer-active animals oxidized pyruvate and beta -hydroxybutyrate at higher rates than those from torpid animals, and flux through complex 4 of the electron transport chain was about three- and fivefold higher than flux through complexes 2-4 and complexes 1-4, respectively. In the hibernating and arousing animals there was no difference in flux through complexes 2-4 and complex 4, suggesting a downregulation of cytochrome c oxidase in liver mitochondria during the hibernation season. Muscle mitochondrial respiration did not differ between the torpid and summer-active states in any of the parameters measured. The data support a regulated, reversible decrease of liver (but not muscle) mitochondrial oxidative phosphorylation in hibernating ground squirrels.     

Opioids and hibernation. II. Effects of kappa opioid U69593 on induction of hibernation in summer-active ground squirrels by "hibernation induction trigger" (HIT)

A "Hibernation Induction Trigger" (HIT) isolated from plasma of winter-hibernating woodchucks induced hibernation in summer-active ground squirrels (Citellus tridecemlineatus). Effects of kappa opioid U69593 on the HIT-induced hibernation were examined. U69593 alone did not elicit marked behavioral alteration or hibernation in summer-active ground squirrels. U69593, however, antagonized hibernation induced by HIT in summer active ground squirrels. In the guinea pig ileum myenteric plexus-longitudinal muscle preparation, woodchuck HIT depressed the electrically-induced contraction. The depression was, however, neither reversed nor blocked by naloxone even when naloxone was used at high doses. This study demonstrates that kappa opioid, at least in the case of U69593, was unable to induce hibernation in the summer-active ground squirrels. The results also demonstrate that woodchuck HIT, like the bear HIT, did not act directly at opioid receptors. Together with our previous observation that naloxone blocked summer hibernation induced by HIT (Bruce et al., Life Sci.., this issue), it is tempting to suggest that HIT may not mediate its effects through kappa opioid receptors but may do so through other types of opioid receptors such as mu or delta. U69593 may antagonize HIT-induced hibernation as a mu or delta receptor antagonist.     

Role of lipids in the assembly of endoplasmic reticulum and dictyosomes in neuronal cells from the cerebral cortex of Yakutian ground squirrel (Citellus undulatus) during hibernation

Phospholipids and cholesterol were assayed in homogenates and microsomal fractions from the cerebral cortex of summer-active, winter-torpid, and winter-active Yakutian ground squirrels (Citellus undulatus). Ultrastructural analysis of both microsomal fraction and intact neurons was performed by serial ultramicrotomy. The levels of sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) were decreased in homogenates from the cerebral cortex of winter ground squirrels compared with the summer-active animals, while the levels of phosphatidylcholine (PC) and cardiolipin (CL) were increased. The level of cholesterol was decreased in the cerebral cortex of winter-torpid animals compared with both winter-active and summer-active animals, and the level of total phospholipids was decreased in comparison to the summer-active animals. Three-dimensional reconstruction of serial membrane profiles displayed the microsomal fraction to be an interconnected system of cisterns and vesicles, which corresponds to endoplasmic reticulum and dictyosomes (Golgi stacks) of intact neurons. In winter the content of PC was increased in the microsomal fraction, while the contents of lysophosphatidylcholine (LPC), PS, phosphatidylinositol (PI), and SM were decreased. In winter-torpid animals compared with the winter-active ones the contents of total phospholipids, PEA, LPC, and cholesterol were decreased. As for the winter-active ground squirrels, their lipid contents did not differ from those in the summer-active animals, but LPC content was decreased. The changes in microsomal lipid contents in intact pyramidal neurons throughout the hibernation were accompanied by disassembly of dictyosomes and endoplasmic reticulum (ER), including the decomposition of polyribosomes to monosomes. The ultrastructural analysis of nucleoli, ER, and dictyosomes of both winter-active and torpid ground squirrels showed a direct correlation between the increasing contents of both cholesterol and total phospholipids (mainly PEA and LPC) in microsomes and the structural recovery of endoplasmic reticulum, Golgi stacks, and nucleoli in intact pyramidal neurons. A role of seasonal variations in lipid contents of brain cells in their adaptation to low temperature is discussed. We also propose an involvement of cholesterol in the activation of protein-synthesizing function of endoplasmic reticulum and Golgi stacks in intact neurons.     

Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5,000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3.5 to 10.0 resulting in protein components having isoelectric points (pIs) of 4.5, 5.2, 5.5, 6.3, and 7.0. Fraction I (comprised of proteins having pIs of 4.5 and 5.2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5.5) and Fraction III (pI 6.3 and 7.0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation. Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3.5 to 6.0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.     

Further studies on opioids and hibernation: delta opioid receptor ligand selectively induced hibernation in summer-active ground squirrels

To examine the possible involvement of multiple opioid receptors in animal hibernation, we infused opioids selective for mu, kappa, and delta opioid receptors into summer-active ground squirrels (Citellus tridecemlineatus). The effects of those opioid treatments on the hibernation induced by HIT (Hibernation Induction Trigger) were also examined. Mu opioids morphine (1.50 mg/kg/day) and morphiceptin (0.82 mg/kg/day) and kappa opioid peptide dynorphin A (0.82 mg/kg/day) did not induce hibernation. On the contrary, morphine, morphiceptin and dynorphin A antagonized HIT-induced hibernation in summer-active ground squirrels. Infusion of delta opioid DADLE (D-Ala2-D-Leu5 enkephalin; 1.50 mg/kg/day), however, induced summer hibernation in a manner comparable to that induced by HIT. It is concluded therefore that delta opioid receptor and its ligand may be intimately involved in animal hibernation. In view of the fact that HIT was obtained from winter hibernating animals and might therefore be responsible for natural hibernation, our results also suggest that naturally occurring mu and kappa opioids may play an important role in the arousal state of hibernation.     

Isolation of a Hibernation Inducing Trigger(s) From the Plasma of Hibernating Woodchucks

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5, 000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3. 5 to 10. 0 resulting in protein components having isoelectric points (pis) of 4. 5, 5. 2, 5. 5, 6. 3, and 7. O. Fraction I (comprised of proteins having pis of 4. 5 and 5. 2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5. 5) and Fraction III (pi 6. 3 and 7. 0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation.

Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3. 5 to 6. 0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when Injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.     

Kinetics of the sodium pump in red cells of different temperature sensitivity

Ouabain-sensitive K influx into ground squirrel and guinea pig red cells was measured at 5 and 37 degrees C as a function of external K and internal Na. In both species the external K affinity increases on cooling, being three- and fivefold higher in guinea pig and ground squirrel, respectively, at 5 than at 37 degrees C. Internal Na affinity also increased on cooling, by about the same extent. The effect of internal Na on ouabain-sensitive K influx in guinea pig cells fits a cubic Michaelis-Menten-type equation, but in ground squirrel cells this was true only at high [Na]i. There was still significant ouabain-sensitive K influx at low [Na]i. Ouabain-binding experiments indicated around 800 sites/cell for guinea pig and Columbian ground squirrel erythrocytes, and 280 sites/cell for thirteen-lined ground squirrel cells. There was no significant difference in ouabain bound per cell at 37 and 5 degrees C. Calculated turnover numbers for Columbian and thirteen-lined ground squirrel and guinea pig red cell sodium pumps at 37 degrees C were about equal, being 77-100 and 100-129 s-1, respectively. At 5 degrees C red cells from ground squirrels performed significantly better, the turnover numbers being 1.0-2.3 s-1 compared with 0.42-0.47 s-1 for erythrocytes of guinea pig. The results do not accord with a hypothesis that cold-sensitive Na pumps are blocked in one predominant form.     

Ca<Superscript>2+</Superscript> responses induced by adrenergic agonists in preadipocytes of mouse and ground squirrel (<Emphasis Type="Italic">Spermophillus undulates</Emphasis>)

Brown adipose tissue of small animals in the cold is able to increase several fold its mass and ability for non-shivering thermogenesis. Adrenergic agonists play a crucial role in the stimulation of the tissue hyperplasia and thermoregulation. In this work, a comparative study of the Ca²⁺ signaling in mouse and ground squirrel brown preadipocytes was carried out. The goal of this study was to elucidate what features of the Ca²⁺ signaling in ground squirrel preadipocytes allow the hibernators to control brown fat hyperplasia in the absence of cold stress. This knowledge would enable us to find ways to control human body mass and to treat diabetes mellitus type two. Low concentrations (0.3–3 μM for mouse cells and 1–10 μM for ground squirrel cells) of the selective α₁- and β-adrenergic agonists cirazoline and isoproterenol, respectively, induced slow Ca²⁺ responses with linear kinetics in brown preadipocytes of both species. High concentrations of the agonists (10–500 μM) caused Ca²⁺ responses with exponential kinetics in ground squirrel brown preadipocytes and suppressed the responses in mouse preadipocytes. Dose-response curves for the agonists were bell-shaped for both mouse and ground squirrel preadipocytes. It should be noted that in preadipocytes of both species β-adrenergic agonist induced stronger responses than α₁-adrenergic agonist did. On the other hand, the responses evoked in ground squirrel brown preadipocytes by both agonists were two orders of magnitude stronger than the responses in mouse preadipocytes. Treatment of the cells with forskolin demonstrated that brown preadipocytes of a ground squirrel had a strong positive feedback in Ca²⁺ signaling, whereas mouse preadipocytes had a negative feedback. The difference found in the preadipocyte Ca²⁺ signaling may explain the different strategies employed in the two species for the regulation of their body fat mass and survival in the cold.     

Ventilatory responses of the ground squirrel, Spermophilus tridecemlineatus, to various levels of hypoxia

1. Minute ventilation (VE) in the semifossorial ground squirrel (Spermophilus tridecemlineatus) increased with increased levels of hypoxia. 2. The increase in VE was brought about primarily by an increase in breathing frequency (f). There was no significant change in tidal volume (VT). 3. The PiO2 threshold for the ventilatory response to hypoxia and position of the ventilatory response curve in the ground squirrel were closer to the semifossorial echidna (Tachyglossus aculeatus) than the completely fossorial mole rate (Spalax ehrenbergi); both the ground squirrel and echidna had a higher PiO2 threshold than the mole rat. 4. The ventilatory response curve was shifted to the left in the mole rat. 5. These observations indicate that the mole rat is the least responsive to hypoxia of the three species.     

The European ground squirrel increases diversity and structural complexity of grasslands in the Western Carpathians

Disturbances are important natural factors affecting biological diversity, community composition, and ecosystem structure. The European ground squirrel is a semi-fossorial organism, and through disturbances caused by burrowing activities, it can play an important role as an ecosystem engineer of grasslands in central and south-eastern Europe. The aim of this study was to assess the response of grassland vegetation to disturbances by the European ground squirrel. We conducted a pairwise survey within a 1-ha study site with homogenous environmental conditions. We compared the vegetation characteristics of 2?×?2-m plots placed on 30 mounds, with paired control plots situated at a distance of 10 m from each mound. The results showed that plots disturbed by the European ground squirrel achieved a higher species richness and diversity and a distinct species composition compared to the undisturbed control plots. Vertical structure of vegetation was also significantly different with a higher proportion of the high and medium vegetation layers on the mounds. Shifts in the composition of plant life forms and life strategies were reflected by the reduction of graminoids and plant competitors, and support of forbs on the mounds. These findings suggest that the European ground squirrel helps to maintain heterogeneity in grassland ecosystems and creates patches of higher diversity and higher structural complexity in the relatively homogenous grassland vegetation of the Western Carpathians.     

Spontaneous adenocarcinoma immunoreactive to cyclooxygenase-2 and transforming growth factor-beta1 in the buccal salivary gland of a Richardson's ground squirrel (Spermophilus richardsonii).

The ground squirrel is used as an experimental animal because of its unique biological nature. A 3-year-old female Richardson's ground squirrel developed a mass, 1.5 cm in diameter, in the buccal mucosa. The mass consisted of neoplastic epithelial cells showing acinar, ductular, intraductal papillary, solid, and lobular growth patterns; the cells were immunoreactive to cytokeratin, cyclooxygenase-2 (a marker of malignancy) and TGF-beta1. After resection, the tumor recurred with increased area having a solid or lobular pattern with little differentiation. This tumor was diagnosed as an adenocarcinoma arising from the buccal gland, the first case reported in the ground squirrel. A prominent desmoplastic reaction was present. The interstitial cells reacted to alpha-smooth muscle actin and vimentin, indicating a myofibroblastic nature, presumably induced by epithelial TGF-beta1.     

A reinvestigation of hemoglobin alterations in ground squirrels while in various hibernation activity states

Characterization of the hemoglobins of winter-hibernating, winter-active and summer-active Arctic ground squirrels (Citellus undulatus) by citrate agar electrophoresis and isoelectric focusing (IEF), pH 5.5-8.5, showed no differences in hemoglobin electrophoretic patterns. Previous studies showing alterations in hemoglobins were most likely the result of artifacts due to the use of whole blood. The Arctic ground squirrel's hemoglobin amino terminal sequence was determined for each activity state and was identical in all cases.     

Morphometric and metabolic indices of disuse in muscles of hibernating ground squirrels.

1. Morphological, biochemical and metabolic characteristics of hindlimb muscles from summer-active (SA), winter-active (WA) and hibernating (H) golden-mantled ground squirrels (Spermophilus lateralis) were examined to identify alterations resulting from seasonal periods of inactivity. 2. Cross-sectional areas of fibers from the soleus were reduced in both WA and H, although only significantly (P less than 0.05) in WA. Fibers in the EDL exhibited significant reductions in cross-sectional areas in both H and WA groups. Muscle fiber and capillary densities were altered in quantitative agreement with changes in cross-sectional areas. 3. Protein content was reduced 20% (P less than 0.05) in EDL from H and WA groups, but reductions (10%) in the soleus were not statistically significant. RNA content in WA and H groups was significantly decreased in soleus (20%) and EDL (35%) compared with SA, but DNA content was unchanged. 4. In the plantaris, triglyceride content was unchanged, but citrate synthase activity in H (210 +/- 13 mumol min-1 g-1) was significantly greater than in SA (177 +/- 10). In contrast, LDH activity in H was reduced by 25% (P less than 0.05) compared with SA. 5. These results demonstrate atrophic effects associated with seasonal inactivity in hibernating ground squirrels, but suggest the existence of natural mechanisms which limit the response.     

The role of succinate dehydrogenase and oxaloacetate in metabolic suppression during hibernation and arousal

Hibernation elicits a major reduction in whole-animal O₂ consumption that corresponds with active suppression of liver mitochondrial electron transport capacity at, or downstream of, succinate dehydrogenase (SDH). During arousal from the torpor phase of hibernation this suppression is reversed and metabolic rates rise dramatically. In this study, we used the 13-lined ground squirrel (Ictidomys tridecemlineatus) to assess isolated liver mitochondrial respiration during the torpor phase of hibernation and various stages of arousal to elucidate a potential role of SDH in metabolic suppression. State 3 and state 4 respiration rates were seven- and threefold lower in torpor compared with the summer-active and interbout euthermic states. Respiration rates increased during arousal so that when body temperature reached 30°C in late arousal, state 3 and state 4 respiration were 3.3- and 1.8-fold greater than during torpor, respectively. SDH activity was 72% higher in interbout euthermia than in torpor. Pre-incubating with isocitrate [to alleviate oxaloacetate (OAA) inhibition] increased state 3 respiration rate during torpor by 91%, but this rate was still fourfold lower than that measured in interbout euthermia. Isocitrate pre-incubation also eliminated differences in SDH activity among hibernation bout stages. OAA concentration correlated negatively with both respiration rates and SDH activity. These data suggest that OAA reversibly inhibits SDH in torpor, but cannot fully account for the drastic metabolic suppression observed during this hibernation phase.     

Smooth muscle contractility and calcium channel density in hibernating and nonhibernating animals

Hibernating animals consistently survive prolonged periods of cold with body temperatures near the freezing point. Previous studies have suggested that regulation of calcium influx may be a fundamental cellular mechanism for cold tolerance in hibernating species. The present study was undertaken to compare (i) the calcium dependence of contractility and (ii) [3H]nitrendipine binding in homogenates of ileal longitudinal smooth muscle from the nonhibernating guinea pig (Cavia porcellus) and a hibernator, the ground squirrel (Spermophilus richardsonii). The contractility studies indicate that both the activation threshold for calcium and the concentration-response curve were shifted to the right in ground squirrel when compared with guinea pig. The binding site density in ground squirrel muscle was about an order of magnitude less than in guinea pig (Bmax = 10 +/- 2 (n = 12) and 86 +/- 6 fmol/mg protein (n = 5), respectively). These results indicate that ground squirrel tissues are less sensitive to external calcium and clearly have fewer calcium channels than the smooth muscle of the non-hibernator. The results continue to support the hypothesis that cold tolerance in hibernating species involves calcium homeostatic control mechanisms.