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. 2002 Apr 1;540(Pt 1):285–294. doi: 10.1113/jphysiol.2001.013188

Reversible impairment of endothelium-dependent relaxation in golden hamster carotid arteries during hibernation

Hideki Saito 1, Sharada Thapaliya 1, Hayato Matsuyama 1, Masakazu Nishimura 1, Toshihiro Unno 1, Seiichi Komori 1, Tadashi Takewaki 1
PMCID: PMC2290212  PMID: 11927687

Abstract

The effects of hibernation on endothelium-dependent vasodilatation were investigated in the golden hamster carotid artery, paying special attention to hibernating body temperature (10 °C). To record mechanical and electrical membrane responses, we applied pharmacological (organ bath) and electrophysiological (microelectrode) techniques, using acetylcholine (ACh; 0.001–100 μm) and ATP (0.01–1000 μm) for endothelium-dependent vasodilatation and sodium nitroprusside (SNP; 0.05–10 μm) for endothelium-independent vasodilatation. At 34 °C, ACh, ATP and SNP each induced a relaxation or a hyperpolarization, and these responses were similar in all the preparations from control and hibernated animals. At 10 °C, on the other hand, ACh-induced relaxations and hyperpolarizations were reduced to approximately 35 % and 50 % of the euthermic level in controls and 1 % and 4 % of the euthermic level in hibernated animals, respectively. In contrast, at 10 °C, ATP induced only a contraction or depolarization in all preparations with no significant difference between control and hibernated animals. SNP-induced relaxations and hyperpolarizations obtained at 34 °C were not attenuated by cooling to 10 °C. In the presence of a P2X receptor blocker, pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 5 μm), at 34 °C ATP-induced relaxations and hyperpolarizations were significantly enhanced whereas no responses were induced by ATP at 10 °C. After endothelium removal, on the other hand, ATP induced only a contraction or depolarization at both 34 °C and 10 °C. These results suggest that depression of endothelium-dependent vasodilator responses to ACh and ATP may occur in the hibernating golden hamster carotid artery.

During hibernation - an unusual physiological phenomenon characterized by hypothermia, hypoxia or ischaemia - animals undergo a dramatic drop in body temperature, a marked decrease in blood pressure, and a fall in heart and respiratory rates (Lyman, 1965; Nedergaad & Cannon, 1990; Nürnberger, 1995). Despite this decline, the peripheral vascular resistance increases with deepening hibernation to keep blood pressure within a reasonable range; it is thought that the enhancement of sympathetic mediation is one of the major contributory factors for this regulation (Lyman & O'Brien, 1963; Ralevic et al. 1997; Karoon et al. 1998; Saito et al. 2001a). It is now widely accepted that dual regulation by sympathetic perivascular nerves and endothelial cells plays an important role in the control of peripheral circulation under various physiological conditions, such as changes in haemodynamic forces, ischaemia and/or hypoxia (Furchgott & Zawadzki, 1980; Kennedy et al. 1985; Palmer et al. 1987; Burnstock, 1988; Ralevic et al. 1990; Rui & Cai, 1991; Milner et al. 1992; Thapaliya et al. 1999).

A number of cryobiological and surgical studies, on the other hand, indicate that endothelial vasodilator function may alter considerably during cooling or long-term cryopreservation for organ transplantation, some being enhanced or unchanged, while others become irreversibly dysfunctional (Monge et al. 1993; García-Villalón et al. 1995; Almassi et al. 1996; Stanke et al. 1998; Smith et al. 1999). Furthermore, there seems to be a material difference in dysfunction rate and irreversibility between the endothelium-dependent and endothelium-independent relaxations (Ingemansson et al. 1996; Murphy et al. 1997; Alexander et al. 1999).

Despite the dramatic adaptation that occurs in the peripheral circulation during hibernation, surprisingly little has been reported about endothelium control from a cryobiological point of view. Thus, the purpose of the present study is to examine whether endothelial vasodilator function is altered during hibernation. To this end, isolated carotid artery rings of the hibernating golden hamster were used to evaluate the endothelium-dependent and -independent vasodilatation to typical vasodilator agents, paying special attention to hibernating body temperature in the experimental design. A preliminary account of these data has been presented previously (Saito et al. 2001b).

METHODS

Animals

Golden hamsters (Mesocricetus auratus) of either sex from our closed colony weighing 120–150 g were used in these investigations. These animals were divided into two groups: one group was left to hibernate for 8–10 weeks and were killed while undergoing the fifth successive bout of deep hibernation for at least 48 h. The other group - control animals of the same age (5–7 months) as those that had undergone hibernation - was maintained in an ambient temperature of 22–24 °C.

Acclimatization for hibernation

Hibernation was induced by manipulating photoperiod and temperature, as described previously (Saito et al. 2001a). In brief, animals for hibernation were placed in a ventilated, refrigerated incubator initially set at 17 °C and with a light:dark photoperiod of 8:16 h. The temperature was gradually reduced by 1 °C per day, together with a reduction of 30 min of light per day, to reach final conditions of an ambient temperature of 5 °C and a photoperiod of 2:22 h. The hamsters were housed individually and nesting material was supplied. Food and water were provided ad libitum. Hibernation was judged by the lack of response to a physical stimulus, such as the sprinkling of sawdust onto the back of the hamster. A sleeping hamster always awoke to this stimulus, whereas a hibernating animal did not. Animals were in hibernation for 8–10 weeks.

In vivo procedures

During a deep hibernation bout of 1–4 days, some animals had their heart beat and respiratory rate monitored. The heart rate of hibernating hamsters was obtained from an electrocardiogram (ECG) recorded using standard electrophysiological techniques via three standard limb leads and a ground lead. Stainless steel electrodes were gently attached to the planta region of corresponding limbs. The respiratory rate of hibernating hamsters was counted visually by the movement of the thorax. These data were compared with those of control and cold-control animals, which were obtained under anaesthesia (sodium pentobarbitone, 50 mg kg−1, i.p.). Rectal temperature from control and hibernating animals was measured using a thermometer (thermistor type PT, Shibaura Electronics, Co., Ltd, Tokyo, Japan), as opportunity permitted.

Experimental temperature

To determine the effect of tissue temperature the investigation was conducted under two separate sets of experimental conditions acclimatized for a normothermic state and a hypothermic state, that is, similar to hibernating body temperature. Each animal group aforementioned was assigned to one of the two sets of conditions for investigations. Each animal was only used for a single experiment at one of these temperatures. The hibernating animals were removed from the cold room and quickly transferred to a thermostatically controlled bath (model LE-100, Advance, Japan) maintained at hibernating body temperature (see the Results section), in which all procedures for the hypothermic experiment were carried out unless stated otherwise.

Mechanical recordings

The hamsters were killed using diethyl ether and exsanguinated in accordance with a protocol approved by the Gifu University Animal Care and Use Committee. The carotid arteries were dissected and cleaned of excess tissue. Ring segments 2–3 mm in length were cut and mounted for isometric tension recording in a 5 ml organ bath, and were superfused at a rate of 3 ml min−1 with physiological salt solution (PSS). The PSS contained (mm): 133 NaCl, 4.7 KCl, 1.3 Na2PO4, 2.5 CaCl2, 0.6 MgSO4, 16 NaHCO3, and 7.8 glucose, gassed with 95 % O2-5 % CO2. Two fine, stainless steel pins, 150 μm in diameter, were introduced through the lumen of the segment. One pin was fixed to the organ bath wall whilst the other was connected to a strain gauge for isometric recording. An initial resting tension of 1 g was applied to the arterial rings, which were consequently left to equilibrate for 70–90 min. At the end of this period the tension on the vessel was taken as the resting tension and no further mechanical adjustment was made during experimentation. Isometric tension was recorded with a transducer (T7-30-240, Orientec, Japan) and displayed on a thermal-array recorder (RTA-1100M, Nihon Kohden).

Responses were assessed in preparations where the tone was raised by a concentration of noradrenaline (NA) that would give 45–55 % of maximal response. The concentration of NA was titrated for each vascular segment by the initial response to 100 μm NA. When the concentration had plateaued, endothelium-dependent vasodilators - ACh, ATP and an endothelium-independent vasodilator, sodium nitroprusside (SNP) - were added in cumulatively increased concentrations to the perfusing solution. After exposure to a drug, the preparation was washed with PSS for at least 15 min before further experimentation. All the responses were expressed as a percentage of the NA-induced contraction. In some cases, to study the relative contribution of mechanical responses elicited by ATP via P2X and P2Y receptors: (1) preparations were pretreated with pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 5 μm), a P2X blocker, for 20 min, or (2) the endothelium was removed by perfusion with warmed PSS containing collagenase (1 mg ml−1) for 15 min into the lumen of the vessels, then preparations being allowed to equilibrate for at least 1 h. The removal of endothelial cells was considered to be successful when no relaxation was elicited by ACh. In other cases, to explore the involvement of prostanoid, nitric oxide (NO) and/or endothelium-derived hyperpolarizing factor (EDHF)-type responses on the vasorelaxant responses elicited by ACh (Doughty et al. 1999; Dora et al. 2000), the preparations were exposed, for at least 20 min before addition of NA, to the following drugs: indomethacin (5 μm), a cyclo-oxygenase inhibitor, Nω-nitro-l-arginine methyl ester (l-NAME; 100 μm), a nitric oxide synthase (NOS) inhibitor, apamin (50 nm), a blocker of small conductance Ca2+-activated K+ channels, and charybdotoxin (ChTX, 50 nm), a blocker of large conductance Ca2+-activated K+ channels. The sensitivities of the preparations to NA were increased in the presence of l-NAME, so the concentration of NA used was reduced in order to achieve a similar increase in tone as in the controls.

Electrophysiological recordings

The isolated artery segments were cut open longitudinally with fine iredectomy scissors and fixed adventitial side down to a rubber base attached to the bottom of a 5 ml experimental chamber. The preparations were perfused at a constant flow rate of 3 ml min−1 with the oxygenated PSS solution gassed with 95 % O2-5 % CO2. Intracellular recordings of membrane potential were made using glass filament microelectrodes filled with 3 m KCl with tip resistances ranging from 60 to 80 MΩ. The electrical activities were monitored on an oscilloscope (CS 4026, Kenwood, Tokyo, Japan) and recorded on a thermal-array recorder (RTA-1100 M, Nihon Kohden, Tokyo, Japan). Impalements were made from the intimal side. Drugs (ACh, ATP and SNP) were applied by addition to the superfused PSS in the required concentrations. After exposure to a drug, the preparation was washed with PSS for at least 15 min before further experimentation. In some cases, as shown above, (1) preparations were pretreated with 5 μm PPADS for 20 min, or (2) the endothelium was removed by superfusion of warmed PSS containing collagenase (1 mg ml−1) for 15 min, then preparations were allowed to equilibrate for at least 1 h. The removal of endothelial cells was considered to be successful when no hyperpolarization was elicited by ACh. In other cases, as also shown above, the preparations were exposed for at least 20 min to the following drugs: indomethacin (5 μm), l-NAME; 100 μm), apamin (50 nm) and charybdotoxin (ChTX, 50 nm).

Drugs

The drugs used (all from Sigma, USA) were as follows: noradrenaline (NA) hydrochloride, adenosine 5′-triphosphate (ATP) disodium salt, acetylcholine chloride (ACh), sodium nitroprusside (SNP), collagenase, pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), indomethacin, Nω-nitro-l-arginine methyl ester (l-NAME), apamin and charybdotoxin (ChTX). NA (0.1 mm) was dissolved in ascorbic acid (0.1 mm). Indomethacin (10 mm) was dissolved in an equimolar concentration of Na2CO3. All the other drugs were dissolved in distilled water. The drugs were serially diluted in PSS to the required final concentrations immediately before the experiments.

Data analysis

All data are expressed as means ± s.e.m. Statistical differences between the groups were determined by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test to see where any significance lay. Student's unpaired t test for group mean comparisons was utilized when only two means were compared. A value of P less than 0.05 was considered statistically significant.

RESULTS

Induction into hibernation

Hamsters commenced hibernation after 6–10 weeks in the cold room and were allowed to hibernate for a further 8 weeks. During this period, bouts of 1–4 days of deep hibernation occurred. There was a significant weight loss in hibernating hamsters compared with the control animals (Table 1). Mean heart and respiratory rate during a bout of hibernation, and the mean rectal body temperature of the hibernated animals, were significantly lower than those of control animals (Table 1).

Table 1.

Body weight, heart and respiratory rate, and body temperature of control and hibernating hamsters

Control Hibernating
Weight (g) 158 ± 4.8 (14) 99 ± 2.7 (8)*
Heart rate (beats min−1) 424 ± 48 (6) 7.8 ± 1.2 (4)*
Respiratory rate (breaths min−1) 92 ± 13 (6) 1.8 ± 0.5 (5)*
Rectal temperature (°C) 34.5 ± 0.2 (8) 9.8 ± 0.6 (6)*
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Data are means ± s.e.m. (number of animals).

*

P < 0.05vs. control.

Experimental temperature

The experimental temperatures were determined according to the mean rectal temperatures of control and hibernated animals, that is 34 °C for the euthermic state and 10 °C for the hypothermic state. It is debatable whether this apparently hypothermic core temperature for an euthermic rodent can be used as a control value. However, although a reasonable assessment remains unclear, based on observations made in other reports, it seems that euthermic body temperature fluctuates considerably between a range of about 33 °C and 36 °C in some hibernators (Waßmer & Wollnik, 1997; Karoon et al. 1998; Ralevic et al. 1998).

Mechanical responses to relaxing factors

The concentrations of NA that gave 45–55 % of the maximal responses were 2 μm and 50 μm for the experiments at 34 °C and 10 °C, respectively.

Acetylcholine

At 34 °C, in all preparations from control and hibernated animals ACh (0.001–100 μm) induced a sustained relaxation in a concentration-dependent manner (Figs 1A, B and 2A). There was no significant difference between the two groups (Fig. 2A). At 10 °C, however, these responses were significantly depressed, and the response in the hibernated preparations was almost negligible (Figs 1A, B and 2A).

Figure 1. Effects of hibernation and/or cooling to hibernating body temperature (10 °C) on mechanical response to acetylcholine (ACh) and ATP in carotid artery of hamster.

Figure 1

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Representative traces showing vasodilator and/or vasoconstrictor response (expressed as increase or decrease in tension) to ACh from a control (A) and a hibernated animal (B) and to ATP from a control animal (C) at 34 °C (left row) and 10 °C (right row), respectively. Preparations were preconstricted by noradrenaline. Vasodilator responses to ACh are markedly attenuated at 10 °C, and the response in a hibernated animal is negligible. At 10 °C, ATP provokes a further contraction.

Figure 2. Mechanical responses to acetylcholine (ACh), ATP and sodium nitroprusside (SNP) in carotid arteries taken from control and hibernated hamsters at 34 °C and 10 °C.

Figure 2

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Concentration-response curves showing vasodilator and/or vasoconstrictor responses to ACh (A), ATP (B) and SNP (C) in isolated arteries taken from control animals at 34 °C (○) and 10 °C (▵), and hibernated animals at 34 °C (□) and 10 °C (•). Vasodilator responses to ACh were markedly attenuated at 10 °C, and the response in hibernated animals was almost negligible. At 10 °C, ATP provokes vasoconstrictions. All results are expressed as means ± s.e.m.n = 6–8. *P < 0.05vs. control at 34 °C.

ATP

ATP (0.01–1000 μm) induced transient relaxations in a concentration-dependent manner at 34 °C in all preparations, and there was no significant difference between the two groups (Fig. 1C and Fig. 2B). However, at 10 °C ATP (1–1000 μm) provoked further contractions in all precontracted preparations, and these responses did not differ between control and hibernated animals (Fig. 1C and Fig. 2B).

Sodium nitroprusside

Sodium nitroprusside (SNP, 0.05–10 μm) mediated concentration-dependent relaxations at both 34 °C and 10 °C, and the responses at 10 °C were similar to those obtained at 34 °C (Fig. 2C). On comparing control and hibernated animals, neither the responses at 34 °C nor those at 10 °C differed significantly (Fig. 2C).

In the presence of PPADS (5 μm), at 34 °C, ATP (0.01- 1000 μm) elicited concentration-dependent relaxations, and the response was enhanced significantly (Fig. 3A). However, no responses were induced at 10 °C by ATP in the presence of PPADS (Fig. 3A). In collagenase-treated preparations which were preconstricted by NA, at 34 °C ATP (0.01–1000 μm) induced further contractions at both 34 °C and 10 °C, and these responses were similar to those from the preparations with endothelium at 10 °C (Fig. 3B). In the presence of indomethacin (5 μm) alone or with l-NAME (100 μm), the relaxation in response to ACh was unaffected at 34 °C and 10 °C, respectively (Fig. 4A and B). Similarly, in the presence of both indomethacin and l-NAME the subsequent addition of apamin (50 nm) in combination with ChTX (50 nm) did not affect the response to ACh, both at 34 °C and 10 °C (Fig. 4C).

Figure 3. Effects of pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and endothelium removal on mechanical responses to ATP in the carotid artery taken from control hamsters at 34 °C and 10 °C.

Figure 3

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A, concentration-response curves showing mechanical responses to ATP in the absence (○) and in the presence of PPADS (5 μm) at 34 °C (□), and in the absence (▵) and in the presence of the same at 10 °C (•). In the presence of PPADS, relaxations evoked by ATP were enhanced at 34 °C while no responses were observed at 10 °C. B, concentration-response curves showing mechanical responses to ATP with (○) and without endothelium at 34 °C (□), and with (▵) and without at 10 °C (•). Without endothelium, ATP provoked further contractions even at 34 °C. Preparations were preconstricted by noradrenaline. Endothelium was removed by perfusion of PSS containing collagenase (1 mg ml−1). All results are expressed as means ± s.e.m.n = 5–8. *P < 0.05vs. control at 34 °C. †P < 0.05vs. control at 10 °C.

Figure 4. Effects of indomethacin, Nω-nitro-l-arginine methyl ester (l-NAME), and a combination of apamin and charybdotoxin on vasodilatory response to acetylcholine (ACh) in the carotid artery taken from control hamsters at 34 °C and 10 °C.

Figure 4

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A, concentration-response curves showing vasodilator responses to ACh in the absence (○) and in the presence of indomethacin (5 μm) at 34 °C (□), and in the absence (▵) and in the presence of the same at 10 °C (•). B, concentration-response curves showing vasodilator responses to ACh in the absence (○) and in the presence of indomethacin (5 μm) + l-NAME (100 μm) at 34 °C (□), and in the absence (▵) and in the presence of the same agents at 10 °C (•). C, concentration-response curves showing vasodilator responses to ACh in the absence (○) and in the presence of indomethacin (5 μm) + l-NAME (100 μm) + apamin (50 nm) + ChTX (50 nm) at 34 °C (□), and in the absence (▵) and in the presence of the same agents at 10 °C (•). All results are expressed as means ± s.e.m.n = 5–6.

Electrical responses to hyperpolarizing factors

At 34 °C and 10 °C, the mean values of the resting membrane potential of carotid arteries did not significantly differ between control and hibernated animals (P > 0.05): the resting membrane potentials were −64.5 ± 0.5 mV (n = 14), −62.5 ± 6.1 mV (n = 8), −59.0 ± 2.2 mV (n = 8) and −55.5 ± 8.3 mV (n = 7) in control and hibernated animals at 34 °C, and control and hibernated animals at 10 °C, respectively.

Acetylcholine

At 34 °C in all preparations from control and hibernated animals, ACh (0.001–10 μm) induced a hyperpolarization in a concentration-dependent manner (Figs 5A, B and 6A). There was no significant difference between the two groups (Fig. 6A). At 10 °C, however, these responses were significantly depressed, and the response in the hibernated preparations was almost negligible (Fig. 5B and Fig. 6A).

Figure 5. Effects of hibernation and/or cooling to hibernating body temperature (10 °C) on electrical membrane response to acetylcholine (ACh) and ATP in carotid artery of hamster.

Figure 5

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Representative traces showing hyperpolarization and/or depolarization response (expressed as change in membrane potential) to ACh from a control (A) and a hibernated animal (B) and to ATP from a control animal (C) at 34 °C (left row) and 10 °C (right row), respectively. All microelectrode impalements were made from the intimal side. Hyperpolarizations to ACh are markedly attenuated at 10 °C, and the response in a hibernated animal is negligible. At 10 °C, ATP provokes a depolarization.

Figure 6. Hyperpolarization or depolarization in response to acetylcholine (ACh), ATP and sodium nitroprusside (SNP) in carotid arteries taken from control and hibernated hamsters at 34 °C and 10 °C.

Figure 6

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Concentration-response curves showing hyperpolarizing and/or depolarizing responses to ACh (A), ATP (B) and SNP (C) in isolated arteries taken from control animals at 34 °C (○) and 10 °C (▵), and hibernated animals at 34 °C (□) and 10 °C (•). Hyperpolarization to ACh was markedly attenuated at 10 °C, and the response in hibernated animals was almost negligible. At 10 °C, ATP provokes depolarizations. All results are expressed as means ± s.e.m.n = 7–8. *P < 0.05vs. control at 34 °C.

ATP

ATP (0.01–1000 μm) induced a hyperpolarization in a concentration-dependent manner at 34 °C in all preparations, and there was no significant difference between the two groups (Fig. 6B). However, at 10 °C ATP (0.1–1000 μm) provoked a depolarization in all preparations, and the responses did not differ between control and hibernated animals (Fig. 5C and Fig. 6B).

Sodium nitroprusside

Sodium nitroprusside (SNP, 0.05–10 μm) mediated a concentration-dependent hyperpolarization at both 34 °C and 10 °C, and the responses at 10 °C were similar to those obtained at 34 °C (Fig. 6C). On comparing control and hibernated animals, neither the responses at 34 °C nor at 10 °C differed significantly (Fig. 6C).

In the presence of PPADS (5 μm), at 34 °C ATP (0.01–1000 μm) elicited a hyperpolarization, and the response was enhanced significantly (Fig. 7A). At 10 °C, however, ATP induced no response in the presence of PPADS (Fig. 7A). In collagenase-treated preparations, ATP (0.01–1000 μm) induced only a depolarization, at both 34 °C and 10 °C (Fig. 7B), and these responses were similar to those from the preparations with endothelium at 10 °C (Fig. 7B). In the presence of indomethacin (5 μm) alone or with l-NAME (100 μm), the hyperpolarization in response to ACh was unaffected at 34 °C and 10 °C, respectively (Fig. 8A and B). Similarly, in the presence of both indomethacin and l-NAME the subsequent addition of apamin (50 nm) in combination with ChTX (50 nm) did not affect the response to ACh at both 34 °C and at 10 °C (Fig. 8C).

Figure 7. Effects of pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and endothelium removal on electrical membrane responses to ATP in the carotid artery taken from control hamsters at 34 °C and 10 °C.

Figure 7

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A, concentration-response curves showing electrical membrane responses to ATP in the absence (○) and in the presence of PPADS (5 μm) at 34 °C (□), and in the absence (▵) and in the presence of the same at 10 °C (•). In the presence of PPADS, hyperpolarization evoked by ATP was enhanced at 34 °C while no responses were observed at 10 °C. B, concentration-response curves showing electrical membrane responses to ATP with (○) and without endothelium at 34 °C (□), and with (▵) and without at 10 °C (•). Without endothelium, ATP provoked depolarization at 34 °C. Endothelium was removed by perfusion of PSS containing collagenase (1 mg ml−1). All results are expressed as means ± s.e.m.n = 5–6. *P < 0.05vs. control at 34 °C. †P < 0.05vs. control at 10 °C.

Figure 8. Effects of indomethacin, Nω-nitro-l-arginine methyl ester (l-NAME), and a combination of apamin and charybdotoxin on hyperpolarizing response to acetylcholine (ACh) in the carotid artery taken from control hamsters at 34 °C and 10 °C.

Figure 8

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A, concentration-response curves showing hyperpolarizing responses to ACh in the absence (○) and in the presence of indomethacin (5 μm) at 34 °C (□), and in the absence (▵) and in the presence of the same at 10 °C (•). B, concentration-response curves showing hyperpolarizing responses to ACh in the absence (○) and in the presence of indomethacin (5 μm) + l-NAME (100 μm) at 34 °C (□), and in the absence (▵) and in the presence of the same agents at 10 °C (•). C, concentration-response curves showing hyperpolarizing responses to ACh in the absence (○) and in the presence of indomethacin (5 μm) + l-NAME (100 μm) + apamin (50 nm) + ChTX (50 nm) at 34 °C (□), and in the absence (▵) and in the presence of the same agents at 10 °C (•). All results are expressed as means ± s.e.m.n = 5–6.

DISCUSSION

In the present work we have studied the effects of hibernation and cooling to hibernating body temperature (10 °C) on endothelium-dependent relaxations in the golden hamster carotid artery. Our findings strongly suggest that during hibernation endothelium-dependent vasodilator dysfunction may occur, whereas the dilator function of the smooth muscle per se may potentially be maintained.

A number of studies on the hibernating golden hamster have shown that the dilator responses of femoral and mesenteric arteries to exogenous ACh remain unchanged (Ralevic et al. 1997, 1998; Karoon et al. 1998), this being consistent with our results obtained at 34 °C. However, the respective values obtained at 10 °C from both control and hibernated animals were significantly depressed, and the same was true of values from our electrophysiological findings at 10 °C. Cryobiological studies have shown that cooling augments the reactivity of rabbit ear arteries (cutaneous) to cholinoceptor stimulation by endothelium-mediated mechanisms, while in femoral arteries (non-cutaneous) it did not affect the relaxation to the same stimulation, with an explanation that during cooling an increased release of endothelial NO might occur in cutaneous arteries, but not in non-cutaneous arteries, when stimulated with ACh, and this difference might be due to a specific feature of cutaneous arteries (Monge et al. 1993; García-Villalón et al. 1995). In our findings, however, the endothelial dilatory responses to ACh of the carotid artery (a non-cutaneous artery) were drastically depressed at 10 °C. At present, with no other data available on the dilator responsiveness of the arterial preparations to ACh at 10 °C, we cannot offer an exact explanation for this discrepancy. Nevertheless, neither can we rule out totally the possibility that the state of hibernation or the hibernating body temperature may play a particular role in the depression of the ACh-induced endothelium-dependent relaxation, especially when we observe the marked difference in the depression between control and hibernated animals.

Previous reports dealing with endothelial responses to dilator drugs via P2Y receptors in hibernated animals have shown that the responses to ATP were vasodilatory, with no difference between control and hibernated animals in hamster mesenteric arteries (Ralevic et al. 1997, 1998), and this is consistent with our result at 34 °C. At 10 °C, on the other hand, the mechanical and electrical membrane responses to ATP were contraction and depolarization, respectively, in both groups. The temperature at which an experiment was carried out, and the side of the microelectrode impalements for the electrophysiological study, should be considered carefully. In the present study, the responses to ATP in endothelium-denuded preparations at 34 °C were contraction or depolarization, and this result was similar to that observed in intact preparations at 10 °C. In addition, the further contraction and depolarization elicited by ATP at 10 °C were completely inhibited in the presence of the P2X blocker PPADS. Besides, the depolarizations elicited by ATP at 10 °C from all the intact preparations were obtained via intimal impalement, where the response to ATP would be expected to be a hyperpolarization due to the close proximity of the endothelial cells and smooth muscle cells (Thapaliya et al. 1999). It is, therefore, most likely that the further contraction and depolarization induced by ATP at 10 °C may be due to a reduction in the participation of endothelial P2Y receptors.

Both mechanical and electrical membrane responses to SNP - an NO donor used to assess the receptor-independent function of the preparations - showed no difference between the two groups at 34 °C and 10 °C, respectively, confirming a lack of change in carotid arterial smooth muscle vasodilator and hyperpolarizing function in hibernation.

Endothelial dysfunction of donor-organ vasculature is one of the most critical issues for organ transplantation (Almassi et al. 1996; Alexander et al. 1999). Reversibility of endothelial function after cold-storage preservation may be a crucial point. Based on our results, the endothelium-dependent vasodilator responses to ACh and ATP appear to be preserved functionally after hibernation, since the responses obtained at 34 °C from hibernated animals were similar to those from control animals.

Numerous studies on endothelium-dependent relaxation and/or hyperpolarization indicate that in large conducting arteries, NO may be the predominant influence under normal circumstances, with an endothelium-derived hyperpolarizing factor (EDHF) acting as a secondary system; on the other hand, in small arteries EDHF appears to be a major determinant of vascular calibre, and may therefore be of primary importance in the regulation of vascular resistance (Nilsson et al. 1994; Garland & McPherson, 1992; Garland et al. 1995). However, based on our results obtained in hamster carotid artery, one of the large conducting vessels, the major contributing factor to active relaxation might be an EDHF, which is nitric oxide-independent, prostanoid-independent, apamin-insensitive and ChTX-insensitive since the relaxation as well as the hyperpolarization in response to ACh remained the same in the presence of indomethacin and l-NAME, and apamin in combination with ChTX (Murphy & Brayden, 1995; Doughty et al. 1999; Lacy et al. 2000). Because our aim is to investigate the effects of hibernation on endothelium-dependent relaxation, this study does not answer the question as to which candidate could be involved in this mechanism; however, we suggest that an interesting and complementary issue for further study would be to examine the effects of several other K+ channel blockers, for instance iberiotoxin, a selective inhibitor of the KCa channel (Murphy & Brayden, 1995). Also important and necessary are studies to prove that the phenomenon is not an artefact caused by particular drugs, since there are reports of differences in efficacy between, for instance, inhibitors such as N-nitro-l-arginine (l-NNA) and l-NAME (Thorin et al. 1999; Richter et al. 2000; Walter et al. 2000).

Since the sensitivities of the preparations to NA were increased in the presence of l -NAME (see Methods), we cannot exclude completely the involvement of NO (Hill et al. 1996; Murphy & Brayden, 1995; Dora et al. 2000); however, taken together, it is most likely that in the hamster carotid artery, endogenous NO may have only a small role as a mediator of endothelium-dependent relaxation.

In conclusion, the main findings of this study suggest that in the hibernating golden hamster carotid artery, reversible impairment of endothelium-dependent relaxation may occur, and this might consequently be suitable for maintaining peripheral resistance since main blood pressure as well as blood flow are known to be markedly decreased during hibernation.

Acknowledgments

This work was supported by a Grant-in-Aid (13660297) for Scientific Research from the Japanese Ministry of Education, Science and Culture.

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