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The cold-shock response--a hot topic.

The cold-shock response of Escherichia coli describes a specific pattern of gene expression in response to abrupt shifts to lower temperatures. This pattern includes the induction of cold-shock proteins, synthesis of proteins involved in transcription and translation, and repression of heat-shock proteins. The identified cold-shock proteins are involved in various cellular functions from supercoiling of DNA to initiation of translation. The major cold-shock protein, CspA, has high sequence similarity with three other E. coli proteins--CspB, CspC, and CspD. Using translational lacZ fusions, cspB was found to be cold-shock inducible at the level of transcription like cspA, while cspC and cspD were not. The Csp proteins, which share sequence similarity with other prokaryotic proteins and with the 'cold-shock domain' of eukaryotic Y-box proteins, may have a function in activating transcription or unwinding or masking RNA molecules. Because the cold-shock response can also be induced by the addition of certain inhibitors of translation, it has been proposed that the state of the ribosome is the physiological sensor for the induction. In addition to E. coli, cold-shock proteins have also been found in other prokaryotic and eukaryotic organisms.

Amino Acid Sequence↗

Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis.

Many plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. In Arabidopsis, cold acclimation involves action of the CBF cold response pathway. Key components of the pathway include rapid cold-induced expression of three homologous genes encoding transcriptional activators, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), followed by expression of CBF-targeted genes, the CBF regulon, that increase freezing tolerance. Unlike Arabidopsis, tomato cannot cold acclimate raising the question of whether it has a functional CBF cold response pathway. Here we show that tomato, like Arabidopsis, encodes three CBF homologs, LeCBF1-3 (Lycopersicon esculentum CBF1-3), that are present in tandem array in the genome. Only the tomato LeCBF1 gene, however, was found to be cold-inducible. As is the case for Arabidopsis CBF1-3, transcripts for LeCBF1-3 did accumulate in response to mechanical agitation, but not in response to drought, ABA or high salinity. Constitutive overexpression of LeCBF1 in transgenic Arabidopsis plants induced expression of CBF-targeted genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog of the Arabidopsis CBF1-3 proteins. However, constitutive overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase freezing tolerance. Gene expression studies, including the use of a cDNA microarray representing approximately 8000 tomato genes, identified only four genes that were induced 2.5-fold or more in the LeCBF1 or AtCBF3 overexpressing plants, three of which were putative members of the tomato CBF regulon as they were also upregulated in response to low temperature. Additional experiments indicated that of eight tomato genes that were likely orthologs of Arabidopsis CBF regulon genes, none were responsive to CBF overexpression in tomato. From these results, we conclude that tomato has a complete CBF cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis and appears to be considerably smaller and less diverse in function.

Acclimatization↗

A single amino acid substitution in the Arabidopsis FIERY1/HOS2 protein confers cold signaling specificity and lithium tolerance.

Low temperature induces the expression of many plant genes through undefined signaling pathways. To gain insight into cold signal transduction mechanisms, we isolated Arabidopsis mutants that exhibited altered regulation of low temperature-induced gene expression. One such mutant, hos2, was shown previously to have an enhanced induction of stress-responsive genes by cold, whereas the expression of these genes under osmotic stress or the phytohormone absciscic acid (ABA) treatments was not affected. Here we further define the targets of HOS2 by examining the regulation of upstream cold-specific CBF transcription factor genes. It was found that the transcript levels of CBF2 and CBF3 were significantly higher in hos2 mutant plants than in the wild type under cold treatments, suggesting that HOS2 may act upstream of CBFs. The HOS2 gene was cloned using a map-based strategy. Surprisingly, HOS2 is identical to the FIERY1 gene that we had described previously. FIERY1 is a general negative regulator that controls cold, osmotic stress, and ABA signal transduction and possesses inositol polyphosphate 1-phosphatase activity. The hos2 mutation rendered the HOS2/FIERY1 recombinant protein completely inactive in the cold but did not substantially affect its activity at warm temperatures. Interestingly, the hos2 mutant protein is extremely tolerant to Li+. This study provides a unique example of a single amino acid substitution in a critical regulator that can lead to conditional changes in protein functions and distinct plant phenotypes. The results reinforce the notion that phosphoinositols are important second messengers in cold signal transduction, and shed light on how the diversity of plant tolerance to cold and other abiotic stresses may evolve due to variations in a common molecular switch.

Amino Acid Sequence↗

Two populations of cold-sensitive neurons in rat dorsal root ganglia and their modulation by nerve growth factor.

Cold sensing in mammals is not completely understood, although significant progress has been made recently with the cloning of two cold-activated ion channels, TRPM8 and TRPA1. We have used rat DRG neurons in primary culture and calcium fluorimetry to identify distinct populations of cold-sensitive neurons, which may underlie different functions. Menthol sensitivity clearly separated two classes of cold-responding neurons. One group was menthol-sensitive (MS), was activated at warmer temperatures and responded faster and with a larger increase in intracellular calcium concentration during cooling; the fraction of MS neurons in culture and their cold sensitivity were both increased in the presence of nerve growth factor. Neurons in the menthol-insensitive (MI) group required stronger cooling for activation than MS cells and neither their proportion nor their cold sensitivity were significantly altered by nerve growth factor. The two groups of cold-sensitive neurons also had different pharmacology. A larger fraction of MS cells were capsaicin-sensitive and coexpression of menthol and capsaicin sensitivity was observed in the absence of NGF. MI neurons were not stimulated by the super-cooling agent icilin or by the irritant mustard oil. Taken together these findings support a picture in which TRPM8 is the major player in detecting gentle cooling, while TRPA1 does not seem to be involved in cold sensing by MI neurons, at least in the temperature range between 32 and 12 degrees C.

Animals↗

Changes in the daily rhythm of serum testosterone levels following superior cervical sympathetic ganglionectomy in the cold-exposed rat: the role of the pineal.

The effect superior cervical sympathetic ganglionectomy (Gx) exerted on the daily rhythm of serum testosterone levels was investigated in cold-exposed rats. Rhythmic changes in pineal and pituitary weights were also measured. 1. Exposure to cold (10 degrees C for 72 h) resulted in a significant decrease of serum testosterone level and in an increase of the pineal weight. 2. In neutral ambient temperature (24 degrees C) Gx, 30 d after operation, led to a moderate, statistically insignificant increase of serum testosterone levels and to decreased pineal weights (statistically significant). 3. The reactions provoked by cold exposure were counteracted by Gx. Testosterone levels, as well as the pineal weight, showed no remarkable change in the Gx, cold-exposed animals. 4. These results confirm our assumption that experimental manipulations of the pineal gland can provoke significant changes in the neuroendocrine system only under special loading circumstances, e.g., cold exposure. Sympathetic denervation of the pineal gland counteracts the cold-induced decrease of testosterone levels by counteracting the pineal antigonadotropic activity. 5. The empirical regression curves of the investigated parameters indicate that Gx or cold exposure provide a shift in the upper and lower limits of the daily rhythm. Partly inverted rhythms were also observed. 6. The presented results are discussed in relation to the parallel changes previously described in serum thyroxin, cholesterol, thyrotropin (TSH), and pituitary TSH levels. Thyroidal-gonadal interactions, as well as cold exposure as a stress-generating factor, have been considered in the possible explanation of the data herein reported.

Animals↗

Caspase inhibition prevents the increase in caspase-3, -2, -8 and -9 activity and apoptosis in the cold ischemic mouse kidney.

Prolonged cold ischemic time is a risk factor for the development of delayed graft function. The adverse impact of cold ischemia may be associated with tubular cell death in the kidney. Caspase-3 is a major mediator of apoptotic cell death. We hypothesized that caspase inhibition would reduce apoptosis and other features of cold ischemia. Kidneys of C57BL/6 mice were perfused with cold University of Wisconsin solution containing a pancaspase inhibitor or vehicle via the left ventricle. The contralateral right kidney was used as a control. The left kidney was stored for 48 h at 4 degrees C to produce cold ischemia. Caspase-3 activity was massively (100-fold) increased in cold ischemic kidneys compared with controls. On immunoblot analysis, the processed form of caspase-3 was increased in cold ischemic kidneys compared with controls. The increase in caspase-3 was associated with significantly more renal tubular apoptosis and brush-border injury. In addition, caspase-2, -8 and -9 activities were increased in cold ischemic kidneys. The pancaspase inhibitor prevented the formation of the processed form of caspase-3 and the increase in caspase activity, and reduced apoptosis and brush-border injury. Caspase inhibition may prove useful in kidney preservation.

Animals↗

Suppression of secretagogue-induced amylase secretion in pancreatic acini of cold-exposed rats.

1. The influence of prolonged exposure to a low ambient temperature on the pancreatic exocrine function was investigated in rats. Amylase concentration in acini prepared from rats housed at 5 degrees C for 2 weeks was specifically and dramatically reduced. The enzyme activity relative to acinar DNA concentration was only 48% of that of control acini obtained from rats reared at the thermoneutral temperature of 25 degrees C. Amylase content relative to acinar protein concentration also decreased by 35%. In contrast, acinar trypsinogen content relative to protein concentration increased to 161% of that of control acini. No comparable change was found in the parotid glands. 2. In acini from cold-exposed rats, amylase release in response to cholecystokinin octapeptide or carbamylcholine was simultaneously reduced when expressed relative to acinar DNA or protein concentration and the dose-response curve shifted downward. However, when the secretory responsiveness of amylase was represented as a percentage of initial acinar enzyme content, the observed decrease disappeared and the dose-response curve was unaffected by cold exposure. 3. Secretagogue-stimulated trypsinogen release was augmented in acini from cold-exposed rats when expressed per acinar protein concentration. Conversely, its percentage release was slightly lowered but the release per acinar DNA concentration remained unaltered. 4. Pancreatic insulin concentration normalized to tissue wet weight or protein concentration was unaffected by prolonged cold exposure but the plasma insulin concentration significantly decreased by 45% as compared with that of thermoneutral rats. 5. Chronically administered exogenous insulin partially restored the decreased acinar amylase concentration in cold-exposed rats. The dose-response curve for amylase normalized by acinar DNA or protein content also tended to be shifted upward by insulin treatment. Acinar trypsinogen concentration in these rats was not significantly altered from that in cold-exposed, non-insulin-treated rats. Percentage release of each enzyme was decreased by exogenous insulin. 6. Cold-induced suppression of pancreatic acinar amylase concentration, resulting in a proportional decrease in secretory responsiveness, was confirmed at the cellular level. Insulin deficiency at a low ambient temperature is likely to be a major cause of cold-induced suppression of amylase biosynthesis and its release via an insulin-pancreatic acinar axis.

Amylases↗

Effect of maternal cold exposure on brown adipose tissue and thermogenesis in the neonatal lamb.

1. This study examines the effect of chronic cold exposure during pregnancy, induced by winter shearing twin-bearing ewes 4 weeks before predicted lambing date, on O2 consumption and CO2 production during non-rapid-eye-movement (REM) sleep in lambs maintained for at least 1 h at warm (28-18 degrees C) and cold (14-5 degrees C) ambient temperatures at 1, 4, 14 and 30 days of age. This was combined with measurement of the thermogenic activity (GDP binding to uncoupling protein in mitochondrial preparations) of perirenal adipose tissue from lambs immediately after birth and at 33 days of age. 2. Lambs born from shorn (cold-exposed) ewes were 15% heavier (P < 0.01) and possessed 21% (P < 0.01) more perirenal adipose tissue that contained 40% more protein and mitochondrial protein than unshorn (P < 0.05) controls. Total GDP binding in perirenal adipose tissue was 40% greater (P < 0.05) in lambs born from shorn ewes but there was no difference in lipid content of this tissue between the two groups. 3. At 1 day of age, lambs born from shorn ewes exhibited a 16% higher (P < 0.05) rate of O2 consumption (per kilogram bodyweight) at the warm temperature and a 40% greater metabolic response to the cold ambient temperature. All lambs born from shorn ewes responded to cold exposure without shivering (i.e. via non-shivering thermogenesis) whilst shivering was measured in four out of seven lambs in the unshorn group. These differences had disappeared by 4 days of age as a result of a 25% increased (P < 0.01) rate of O2 consumption in the warm in lambs born from unshorn ewes and a 20% decrease (P < 0.05) in the response to the cold in lambs from shorn ewes. Shivering during cold exposure was measured in six out of nine lambs born from shorn ewes indicating a rapid alteration in thermoregulatory responses to cold during the first few days of life. 4. The levels of GDP binding and mitochondrial protein in perirenal adipose tissue fell by one-third in both groups of lambs during the first 33 days of life whereas lipid content either increased or was unchanged. This indicated that brown adipose tissue (BAT) was developing the characteristics of white adipose tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗

Autonomic control of colonic tone and the cold pressore test.

BACKGROUND: Cardiovascular responses to cold stimulation are well characterised. It is unclear, however, whether cold pain stimulates responses in colonic tone in the transverse and sigmoid regions. AIMS: To assess the effects of cold stimulation on tone nd motility in the transverse and sigmoid colon and on cardiovascular autonomic activity. METHODS: Phasic and tonic motility of the transverse and sigmoid colon, pulse rate, and beat to beat pulse variability (which are measures of centrally mediated changes in autonomic function) were measured before, during, and after a standard cold pressor test in 22 healthy volunteers. RESULTS: Cold pain induced a significant increase in colonic tone but not phasic contractility in the transverse and sigmoid regions. Simultaneously, cold pain increased pulse interval variability. CONCLUSION: The findings are consistent with the hypotheses that cold pain produces coactivation of both the sympathetic and parasympathetic limbs of the autonomic nervous system and that cold induced changes in colonic tone are temporally associated with alterations in central autonomic nervous activity.

Adult↗

Cold periods and coronary events: an analysis of populations worldwide.

STUDY OBJECTIVE: To investigate the association between cold periods and coronary events, and the extent to which climate, sex, age, and previous cardiac history increase risk during cold weather. DESIGN: A hierarchical analyses of populations from the World Health Organisation's MONICA project. SETTING: Twenty four populations from the WHO's MONICA project, a 21 country register made between 1980 and 1995. PATIENTS: People aged 35-64 years who had a coronary event. MAIN RESULTS: Daily rates of coronary events were correlated with the average temperature over the current and previous three days. In cold periods, coronary event rates increased more in populations living in warm climates than in populations living in cold climates, where the increases were slight. The increase was greater in women than in men, especially in warm climates. On average, the odds for women having an event in the cold periods were 1.07 higher than the odds for men (95% posterior interval: 1.03 to 1.11). The effects of cold periods were similar in those with and without a history of a previous myocardial infarction. CONCLUSIONS: Rates of coronary events increased during comparatively cold periods, especially in warm climates. The smaller increases in colder climates suggest that some events in warmer climates are preventable. It is suggested that people living in warm climates, particularly women, should keep warm on cold days.

Adult↗

Heat-related and cold-related deaths in England and Wales: who is at risk?

BACKGROUND: Despite the high burden from exposure to both hot and cold weather each year in England and Wales, there has been relatively little investigation on who is most at risk, resulting in uncertainties in informing government interventions. OBJECTIVE: To determine the subgroups of the population that are most vulnerable to heat-related and cold-related mortality. METHODS: Ecological time-series study of daily mortality in all regions of England and Wales between 1993 and 2003, with postcode linkage of individual deaths to a UK database of all care and nursing homes, and 2001 UK census small-area indicators. RESULTS: A risk of mortality was observed for both heat and cold exposure in all regions, with the strongest heat effects in London and strongest cold effects in the Eastern region. For all regions, a mean relative risk of 1.03 (95% confidence interval (CI) 1.02 to 1.03) was estimated per degree increase above the heat threshold, defined as the 95th centile of the temperature distribution in each region, and 1.06 (95% CI 1.05 to 1.06) per degree decrease below the cold threshold (set at the 5th centile). Elderly people, particularly those in nursing and care homes, were most vulnerable. The greatest risk of heat mortality was observed for respiratory and external causes, and in women, which remained after control for age. Vulnerability to either heat or cold was not modified by deprivation, except in rural populations where cold effects were slightly stronger in more deprived areas. CONCLUSIONS: Interventions to reduce vulnerability to both hot and cold weather should target all elderly people. Specific interventions should also be developed for people in nursing and care homes as heat illness is easily preventable.

Adolescent↗

Skin temperature recovery from cold provocation in workers exposed to vibration: a longitudinal study.

BACKGROUND: Vibration white finger (VWF) is characterised by arterial hyperresponsiveness and vasoconstriction following cold provocation. Several years after of removal from exposure, most subjects show improved finger systolic blood pressure (FSBP) under conditions of cold challenge, but continue to report cold hands and finger blanching. AIMS: To assess the underlying reasons for the persistence of cold symptoms. METHODS: A total of 204 former users of pneumatic tools with cold related hand symptoms were evaluated and then re-evaluated a year later. Symptoms were evaluated using the Stockholm Workshop Scale. Finger systolic blood pressure per cent (FSBP%) was assessed by comparing digital blood pressure in a cold provoked and normalised state. Fingertip skin temperature was measured during cooling and occlusion and during rewarming and recovery. RESULTS: There were dramatic improvements in FSBP% (14.3 mm Hg %), modest improvement in recovered skin temperature (0.86 degrees C), and no change in symptom stage. When the most symptomatic subjects (n = 75) were compared with the less symptomatic subjects (n = 129), there were similar inter-test improvements in FSBP%. Skin temperature recovery improved in the less symptomatic (+1.49 degrees C), but did not change in the most symptomatic group (-0.12 degrees C). However, the more symptomatic group had higher temperatures at the initial test, thus qualifying the result. CONCLUSIONS: Skin temperature recovery after cold challenge in subjects with VWF remains reduced in the symptomatic subjects several years after exposure removal. This is evident even when blood pressure has increased in the setting of cold provocation. Results suggest that in VWF, the dermal circulation remains impaired, even after the restoration of arterial blood pressure in the digits.

Adult↗

Effect of a novel 5-lipoxygenase activating protein inhibitor, BAYx 1005, on asthma induced by cold dry air.

BACKGROUND: Leukotrienes have been implicated in the mediation of airway obstruction induced by hyperventilation of cold dry air in asthmatic subjects. The effect of a novel inhibitor of 5-lipoxygenase activating protein, BAYx 1005, on the bronchospastic response to cold dry air hyperventilation was investigated in asthmatic patients. METHODS: After a screening cold dry air hyperventilation challenge to document cold air responsiveness, 16 asthmatic subjects (baseline forced expiratory volume in one second (FEV1) > 60% of predicted) underwent cold air challenge three hours after receiving 750 mg of BAYx 1005 or placebo using a randomised, double blind, crossover design. Leukotriene synthesis inhibition was estimated by measuring the concentration of leukotriene B4 in whole blood stimulated with calcium ionophore A21387. RESULTS: Treatment with BAYx 1005 produced a 34% (95% CI 11 to 63) increase in the amount of cold air minute ventilation required for a 10% decrease in FEV1 (PD10VE) compared with placebo (mean (SE) 37.6 (1.12) 1/min compared with 28.0 (1.13) 1/min, p < 0.006). The PD20VE increased 19% (95% CI 8 to 31) after treatment with BAYx 1005 compared with placebo (57.3(1.10)1/min versus 48.1 (1.10) 1/min, p < 0.002). Treatment with BAYx 1005 produced a 15.4% decrease in ionophore-stimulated LTB4 production, while treatment with placebo produced a 7.1% increase in ex vivo LTB4 (p < 0.02). CONCLUSIONS: Treatment with BAYx 1005, a novel inhibitor of leukotriene synthesis, produced a significant blunting of cold dry air responsiveness consistent with the hypothesis that leukotrienes mediate part of the bronchoconstriction induced by hyperventilation of cold dry air.

Adolescent↗

Growth of interscapular brown adipose tissue in cold-acclimated hypophysectomized rats maintained on thyroxine and corticosterone.

Brown adipose tissue (BAT) of rats is known to grow in response to acclimation to cold. The growth is accompanied by changes in mitochondrial polypeptide composition (an increase in the relative proportion of a polypeptide of molecular weight 32,000, known to be associated with the thermogenic proton conductance pathway). The mediator of the change in mitochondrial polypeptide composition is unknown. The objective of these experiments was to find out whether any of the pituitary hormones might be the mediator. Treatment of rats with growth hormone failed to alter BAT size or mitochondrial polypeptide composition. BAT grew and the change in BAT mitochondrial polypeptide composition occurred in cold-acclimated hypophysectomized rats, maintained on thyroxine and corticosterone to ensure their survival in the cold. It is concluded that none of the pituitary hormones is the mediator for the cold-induced change in BAT mitochondrial polypeptide composition or is required to exert a direct effect on BAT for cold-induced BAT growth to occur. It also seems unlikely that more than a maintenance amount of glucocorticoids is required for normal cold-induced growth of BAT; these hormones are thus also unlikely to mediate the change in BAT mitochondrial polypeptide composition. The requirement for no more than a maintenance amount of thyroxine for BAT growth and for the cold-induced change in BAT mitochondrial polypeptide composition confirms previous conclusions drawn from studies on cold-acclimated thyroidectomized rats.

Acclimatization↗

Effect of different carbon sources and cold shock on protein synthesis by a psychrotrophic Acinetobacter sp.

The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80.

Acetates↗

Effects of noradrenaline and nicotinic acid on plasma free fatty acids and oxygen consumption in cold-adapted rats.

A 3-h noradrenaline (NA) infusion (1.5 microgram kg-1 min-1) produced a sustained enhanced oxygen consumption (O2 cons.) in cold-adapted rats. Plasma free fatty acid (FFA) levels were elevated by NA in control and in cold-adapted rats, but to lesser extent in cold-adapted rats; the increase was maintained at a plateau in both groups during the entire period of NA infusion. A 1-h nicotinic acid (Nic A) infusion (1.5 mg kg-1 min-1) added to the NA infusion inhibited the calorigenic response to NA in cold-adapted rats and reduced the elevated plasma FFA concentration in control and in cold-adapted rats to values below basal levels. However, when the Nic A infusion was stopped, the O2 cons. was increased again in cold-adapted rats by the uninterrupted NA infusion, without the simultaneous increase of the plasma FFA concentration; the plasma FFA concentration was maintained in cold-adapted rats below basal values and merely brought back to basal levels in control rats. From these results, it is suggested that plasma FFA are not an essential substrate to the calorigenic response to NA observed in cold-adapted rats, as 85% of the response can occur when the plasma FFA concentration is very low.

Adaptation, Physiological↗

Effect of norepinephrine on gluconeogenesis in perfused livers of cold-exposed rats.

The effects of norepinephrine and glucagon on gluconeogenesis were studied in hemoglobin-free perfused liver from rats kept for 1-20 days at 4 degrees C. When rats were starved for 24 h at 4 degrees C, the plasma glucose level of rats exposed to cold for 5, 10, and 20 days was significantly higher than that of rats for 1 day, but hepatic glycogen decreased to the same level in all groups. In the isolated perfused liver, basal rates of oxygen consumption and glucose production increased slightly through 5 days of cold exposure and returned to control levels after 20 days of cold exposure. The rates of glucose production from lactate, pyruvate, sorbitol, and glycerol increased by 20-30% after 5 days of cold exposure. The stimulation of gluconeogenesis from these substrates by norepinephrine and phenylephrine increased markedly at all time periods from 1 to 20 days in the cold, with a maximum at 5 days. The stimulation of glycogenolysis by norepinephrine was not affected by cold exposure. The response to catecholamines decreased markedly in liver perfused with calcium-free medium and/or with phentolamine. The stimulation of gluconeogenesis by glucagon increased only in rats exposed to cold for 20 days. The results obtained suggest that the stimulation of hepatic gluconeogenesis by cold is due to an alpha-adrenergic response, and the activation occurs beyond the interaction of norepinephrine with its receptor.

Acclimatization↗

Different effect of cold storage and rewarming on three pH regulating transporters in isolated rat hepatocytes.

Disturbances in hepatic pH homeostasis are thought to participate in the functional damage to liver grafts caused by the cold preservation and warm reperfusion necessitated by transplantation surgery. We have used an in vitro model of isolated rat hepatocytes suspended in cold University of Wisconsin (UW) solution and subsequently cultured at 37 degrees C to evaluate liver cell pH regulatory mechanisms after cold preservation and rewarming. Cells were kept for up to 72 h in cold UW solution, and at 24-h intervals intracellular pH (pHi) was measured after 60-90 min of warm culture by cytofluorometry using the fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. When challenged with an alkaline load by isohydric HCO(3)(-)-CO(2) steps, hepatocytes exhibited similar maximal pH(i) values and recovered at the same rate, irrespective of cold storage time, indicating that Cl-/HCO(3)- exchange activity is quite resistant to hypothermic storage and subsequent rewarming. In parallel studies, cells were subjected to an acid load by the NH4Cl pulse technique in bicarbonate buffer containing 50 microM ethylisopropylamiloride to block Na+/H+ exchange. Despite similar nadir pH(i) (lowest pH(i) values due to acid load), the subsequent pH(i) recovery rate that reflects Na+-(HCO(3)-)n cotransport activity was increased significantly after hypothermic preservation. Hepatocytes were also perfused with a bicarbonate-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, and Na+/H+ exchange activity was evaluated using the same acid load protocol. Although cells always exhibited similar steady-state initial pH(i) and nadir, the rate of pH(i) recovery decreased significantly as a function of cold storage time in UW solution. Finally, intracellular buffering capacity was calculated from the sudden pH(i) changes induced by HCO(3)(-)-CO(2) steps or NH4Cl pulses and was found to remain stable throughout the 72 h of cold preservation. Therefore, the results strongly suggest that cold preservation and rewarming disturb hepatocellular pH regulatory mechanisms by attenuating Na+/H+ exchange and increasing Na+-(HCO(3)-)n cotransport, whereas Cl-/HCO(3)- exchange is not affected.

Adenosine↗