INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE HYPOTHALAMUS, in which a number of neuropeptides has been demonstrated to stimulate or suppress food intake (6), is considered an important organ for the regulation of appetite and energy homeostasis (2, 13). Recently, a novel hypothalamic peptide family (subsequently termed orexins) was discovered in a cytoplasmic calcium-level assay on several cells expressing individual orphan G protein-coupled receptors (15). Orexins include orexin-A and orexin-B, proteolytically derived from the same precursor protein (15). Intracerebroventricularly administered orexin-A and -B stimulate food consumption in a dose-dependent manner, and their production is affected by the nutritional state of rats (15), but little is known about other physiological roles. The expression pattern of mRNA encoding two orexin receptors (OX₁R and OX₂R) in the hypothalamus (21) supports its proposed role in the regulation of feeding (15), but its central distribution is extensive and markedly differentiated between OX₁R and OX₂R (21). These data indicate additional functions of physiological importance for this novel peptide family.

Several peptides that affect food intake have been shown to have some effects on cardiovascular response and sympathetic nerve activity (5, 18). Within the hypothalamus, orexin nerve fibers (4) and orexin receptors, especially OX₂R (21), are found extensively in the paraventricular nucleus (PVN). PVN neurons project directly to the sympathetic preganglionic neurons in the spinal cord and control sympathetic outflow (19). These findings raise the possibility that orexins affect cardiovascular function through their action on the central nervous system. Because anesthesia is well known to affect the cardiovascular and autonomic nervous systems profoundly (9, 24), the present study used conscious, freely moving rats to investigate the effect of intracerebroventricularly administered orexins on mean arterial pressure (MAP), heart rate (HR), and sympathetic nerve activity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal preparation and data collection. Male Wistar rats weighing 350-450 g each were implanted with a lateral cerebroventricular cannula while under anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg). A 24-gauge stainless steel guide cannula (length 19 mm) was positioned 2.5 mm from the cortex surface and 1 mm above the left lateral cerebroventricle through a burr hole located stereotaxically 0.8 mm posterior and 1.5 mm lateral to the bregma. The guide cannula was fixed to the skull with four screws and dental cement. Acute elevation (>20 mmHg) in MAP and persistent (at least 10 min) water-drinking response to intracerebroventricular administration of 10 pmol ANG II were both considered to be indicators of cannula patency and proper placement in the ventricular system. Approximately 10 days later, the cannulated rats were given pentobarbital sodium anesthesia (50 mg/kg ip). SP-31 tubing heat coupled to an SP-50 and a PE-50 catheter was inserted into the abdominal aorta and the inferior vena cava for measurement of blood pressure (BP) and for intravenous administration of drugs, respectively. The arterial catheter, filled with heparinized (10 U/ml) saline solution, was connected to a Statham pressure transducer (Gould, Saddle Brook, NJ) to monitor BP, and the venous catheter was sealed. HR was monitored with a cardiotachometer (model 1321; San-Ei, Tokyo, Japan) triggered by an electrocardiogram (ECG) signal that was recorded via subcutaneous electrodes implanted into the chest. For the measurement of renal sympathetic nerve activity (RSNA), a left renal nerve bundle was dissected carefully via a retroperitoneal approach and freed from the surrounding tissue under stereoscopic microscopy. The nerve was placed on a bipolar electrode made of Teflon-coated wire (Cooner Wire, Chatsworth, CA) and covered with silicone rubber (Semicosil 902A and B cement; Wacker Chemicals East Asia, Tokyo, Japan). Arterial and venous catheters and electrode leads for recording ECG and RSNA were tunneled under the skin to exteriorize at the nape of the neck. Spike potentials, which were amplified (Biophysioamplifier AVB-9; Nihon Kohden, Tokyo, Japan) and filtered (50 to 1,000 Hz), were monitored on a storage oscilloscope (model VC-9A; Nihon Kohden) and continuously recorded on a magnetic tape recorder (Sony, Tokyo, Japan). Through the window discriminator, impulses were then fed into a pulse counter (MET-1100; Nihon Kohden), and the output was digitized, printed as a histogram, and recorded simultaneously with BP and HR on a thermal rectigraph (San-Ei). Tapes were later played back, and the RSNA waveforms were integrated after full-wave rectification using an amplitude analyzer (series 5500; Concurrent, Fort Lauderdale, FL) with the sample hold function reset to baseline by an internal timer set at 5 s. Absolute values for integrated RSNA were corrected before data analysis by subtracting the residual electrical output (background noise level) recorded from the integrator after an intravenous injection of hexamethonium (20 mg/kg iv). All burstlike activity in the RSNA disappeared completely after the injection of hexamethonium, indicating that the recorded neural activity was the result of efferent, but not afferent, renal nerve fibers.

Measurement of plasma catecholamine. For the measurement of plasma catecholamine (CA), another group of weight- and age-matched rats was anesthetized with pentobarbital sodium (50 mg/kg ip), and arterial and venous catheters were inserted into the abdominal aorta and inferior vena cava, respectively. One milliliter of arterial blood was withdrawn through the cannula in the abdominal aorta. To avoid the effect of acute hemorrhage, the same amount of blood was infused into the intravenous catheter at the same speed as blood was withdrawn. The donor blood was obtained from other conscious, male Wistar rats. These blood samples were immediately heparinized and centrifuged at 3,000 rpm and reconstituted in 0.3 ml of 0.5 M acetic acid solution to obtain the final samples. The CA concentration was measured by high-performance liquid chromatography and electrochemical detection using an Eicompak CA-5ODS column (Eicom, Kyoto, Japan).

Experimental protocol. All experiments were performed on conscious, freely moving rats 1-5 days after surgery. The rats were divided into five groups (n = 8 or 9/group). Chow and water were not available during the recording time. After BP, HR, and RSNA stabilized, 5 µl of vehicle (physiological saline solution) or orexin-A and -B (Peptide Institute, Osaka, Japan; 0.3, 3.0 nmol, respectively) were injected intracerebroventricularly into conscious rats through an infusion cannula (30-gauge stainless steel tubing) connected to a 50-µl microsyringe by an automatic injector (LMS, Tokyo, Japan) at a rate of 2.5 µl/min for 2 min. This injection was given by inserting the infusion cannula 1 mm beyond the tip of the guide cannula. In five different groups of rats (n = 7/group), we administered vehicle or orexin-A and -B (0.3, 3.0 nmol, respectively) intracerebroventricularly and collected blood samples for the measurement of plasma CA. Blood samples were obtained 15 min before, as well as 10 and 60 min after, vehicle or orexin intracerebroventricular administration. After each experiment, Pontamine sky blue (1 µl) was injected to verify the correct placement of the intracerebroventricular cannula tip.

Statistical analysis. All data are expressed as means ± SE, and statistical analyses were performed using ANOVA for repeated measurements followed by a Bonferroni multiple-comparisons test. To provide a description of both the duration and magnitude of the cardiovascular and sympathetic responses, the area under the curve (AUC) was calculated (1). Maximum changes from control values and the AUC were analyzed using a Student's t-test. The correlation coefficient (r) was analyzed using a Pearson's correlation coefficient. P < 0.05 was considered statistically significant.

	RESULTS

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Effects of orexins on MAP, HR, and RSNA. Intracerebroventricularly administered orexin-A resulted in a rapid and progressive increase in MAP, with a 15.3 ± 1.1 mmHg increase at a high dose (3.0 nmol; Fig. 1). HR also increased rapidly and reached peak value 15 min after orexin-A (3.0 nmol) administration. This high dose of orexin-A additionally resulted in an ~60% increase in RSNA 10 min after injection, which persisted for ~15 min. RSNA also increased transiently at a low dose (0.3 nmol) of orexin-A. Increases in all these parameters were significantly larger at 3.0 nmol than at 0.3 nmol. Vehicle (saline; n = 8) did not produce any effects on MAP, HR, or RSNA (Fig. 1). To further clarify the relationship between sympathetic nerve activity and cardiovascular responses, the correlation coefficient (r) between RSNA and MAP or HR was examined. There was a statistically significant correlation between RSNA and MAP (r = 0.69 and r = 0.83, respectively; both P values <0.001) or HR (r = 0.76 and r = 0.89, respectively; both P values <0.001) at 0.3- and 3.0-nmol doses in the orexin-A-injected group. Central orexin-B also produced a significant increase in MAP, with a 10.8 ± 0.2 mmHg increase at the high dose, this response pattern being similar to what was observed for orexin-A administration (Fig. 2). HR also rapidly increased and returned to the control level within 30 min at 3.0 nmol. In contrast to the results with orexin-A, RSNA did not increase significantly at any dose of orexin-B (Fig. 2). Increases in MAP and HR were larger at 3.0 nmol than at 0.3 nmol. For each dose, the maximum changes from control values during recording time (60 min) were compared for orexin-A and -B (Fig. 3A). An increase in MAP induced by central orexin-A was 1.5-fold larger than that of orexin-B for both doses, but significant differences were not observed in HR. The increase in RSNA produced by intracerebroventricularly administered orexin-A was larger than that of orexin-B at 3.0 nmol (Fig. 3 A). The AUC was calculated for the 60-min period immediately after peptide injection for each animal within a group to provide a description of both the duration and magnitude of the cardiovascular and sympathetic responses (Fig. 3B). The AUC in MAP and HR was significantly larger in orexin-A than -B at only 3.0 nmol. On the other hand, the AUC in RSNA was >10-fold larger in orexin-A (2,027.3 ± 383.4 % · m; P < 0.05) than -B (186.0 ± 68.1 % · m) at 3.0 nmol (Fig. 3B). At a low dose (0.3 nmol) of orexin-A, the AUC in RSNA (607.2 ± 118.9 % · m, P < 0.05) was approximately sixfold larger than at the same dose of orexin-B (99.1 ± 51.2 % · m).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Time course of changes in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) during the 60 min after intracerebroventricular administration of orexin-A (0.3, 3.0 nmol) or vehicle (saline) in conscious rats. Vertical dotted line indicates time 0; bpm, beats/min. All data are means ± SE; n is no. of animals. * P < 0.05 vs. vehicle;  P < 0.05 vs. orexin-A (0.3 nmol).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Time course of changes in MAP, HR, and RSNA during the 60 min after intracerebroventricular administration of orexin-B (0.3, 3.0 nmol) or vehicle (saline) in conscious rats. Vertical dotted line indicates time 0 min. All data are mean ± SE; n is no. of animals. * P < 0.05 vs. vehicle;  P < 0.05 vs. orexin-B (0.3 nmol).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Bar graph showing maximal changes () from control values (A) and area under the curve (AUC; B) for MAP, HR, and RSNA during the 60 min after intracerebroventricular administration of orexin-A and -B (0.3, 3.0 nmol, respectively) in conscious rats. All data are means ± SE; n is no. of animals. * P < 0.05 vs. orexin-B for each dose.

Effects of orexins on plasma catecholamine. A high dose of orexin-A (3.0 nmol) increased plasma epinephrine (Epi) from 136.4 ± 9.1 to 433.7 ± 59 pg/ml 10 min after injection (Fig. 4A). A tendency toward an increase in Epi was observed with a high dose of orexin-B, but it was not significant (Fig. 4B). Changes in plasma norepinephrine (NE) concentration did not occur at low doses of orexin-A and -B (Fig. 4, A and B). However, with the high dose of orexin-A, plasma NE increased from 104.5 ± 4.2 to 351.1 ± 34.6 pg/ml 10 min after injection, and the increase continued to 284.7 ± 23.8 pg/ml after 60 min. The magnitude of increase in plasma CA induced by central orexin-A was significantly larger at 3.0 nmol than at 0.3 nmol (Fig. 4A). The high dose of orexin-B produced an increase in plasma NE from 104.1 ± 5.1 to 253.6 ± 54.1 pg/ml 10 min after injection, but the level decreased after 60 min (Fig. 4B). Vehicle (saline; n = 7) did not produce any effects on plasma CA (Fig. 4A).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of intracerebroventricular administration of vehicle (saline) or orexin-A (A; 0.3, 3.0 nmol) and orexin-B (B; 0.3, 3.0 nmol) on plasma concentrations of epinephrine and norepinephrine in conscious rats. 0 min, time of administration. All data are means ± SE; n is no. of animals. * P < 0.05 vs. preadministration values.  P < 0.05 vs. orexin-A (0.3 nmol).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This is the first study showing the effects of central orexins on cardiovascular parameters and sympathetic nerve activity recorded directly in conscious, unrestrained rats. Intracerebroventricular administration of orexin-A (0.3 and 3.0 nmol) resulted in a progressive increase in MAP; this MAP increase is also associated with increases in HR and RSNA. Significant correlations were observed between RSNA and MAP or HR for both doses in orexin-A. Although RSNA does not always reflect general sympathetic nerve activity, it is likely that orexin-A induced increases in MAP and HR due to an increase in sympathetic outflow. Similarly, centrally administered orexin-B increased MAP and HR dose dependently, but there were no significant changes in RSNA at any dose. In the regulation of food intake, no significant differences between the effects of orexin-A and -B have been observed (15). Our study indicated that the responses of cardiovascular and sympathetic nerve activity induced by central orexin-A were larger than those of orexin-B. In almost all orexin intracerebroventricularly administered rats, increases in locomotor activities, such as chewing and grooming, were observed. Muscle exercise and postural change are well known to induce the activation of sympathetic outflow (12). To exclude the effects of locomotion on these parameters, we also injected orexin-A and -B (3.0 nmol; n = 6) centrally in rats anesthetized with pentobarbital sodium (50 mg/kg ip). Intracerebroventricularly administered orexin-A increased MAP (control value: 95.2 ± 0.9 mmHg; maximum value: 105.9 ± 3.2 mmHg; P < 0.05), HR (control value: 359.2 ± 2.6 beats/min; maximum value: 394.3 ± 12.2 beats/min; P < 0.05), and RSNA (control value: 100%; maximum value: 131.1 ± 4.5%; P < 0.05), and orexin-B increased MAP (control value: 93.4 ± 1.5 mmHg; maximum value: 101.8 ± 2.3 mmHg; P < 0.05) and HR (control value: 361.5 ± 4.1 beats/min; maximum value: 388.2 ± 8.7 beats/min; P < 0.05), indicating that the increases in these parameters were not due to the rats' activated locomotion.

The existence of regional differences in sympathetic outflow has been demonstrated (23). Thus, to examine systemic sympathetic outflow induced by central orexin, plasma CA was measured under similar conditions to record nerve activity. High doses of orexin-A and -B increased plasma NE, the effect being larger and lasting longer with orexin-A. Therefore, it is likely that the orexin-induced increase in sympathetic nerve outflow leads to the increase in plasma NE, which produces cardiovascular responses. The elevated circulating levels of Epi, as well as NE after injections of the high dose of orexin-A, suggest the activation of the sympathoadrenomedullary system (SA system). In contrast to orexin-A, central orexin-B did not produce an increase in plasma Epi. The large pressor response induced by central orexin-A, compared with that induced by orexin-B may be due to activation of the SA system in addition to sympathetic outflow. These results suggest that intracerebroventricularly administered orexin-A and -B produce cardiovascular responses via different central mechanisms.

Both orexin-A and -B nerve fibers projected widely into the rat brain (4). mRNA for two orexin receptors (OX₁R and OX₂R) distributed extensively in the rat brain (21). Within the hypothalamus, OX₁R mRNA is most abundant in the ventromedial hypothalamic nucleus (21), which plays an important role in the homeostatic regulation of body metabolism mediated through the sympathetic nerves (17). In contrast, OX₂R mRNA exists mainly in the PVN (21), which is involved in the integration of the autonomic nervous and neuroendocrine systems (19). Orexin-A and -B bind to both receptors, but the affinity for OX₁R and OX₂R is different (15). Although our study could not elucidate the action sites of both orexins, our observation that the effects of orexin-A and -B were quantitatively and qualitatively different suggests that both orexins play an important, but different, role in the central regulation of the autonomic nervous system. Although the pathophysiological role of the activation of sympathetic outflow induced by orexin is not clear, a number of studies have clearly documented the close relationship between obesity, hypertension, and altered cardiovascular responses (10). In this regard, leptin, the peptide product of the obese gene (25), has been reported to decrease food intake (3) and to produce an increase in MAP (5, 16) and RSNA (5). Therefore, these neuropeptides, leptin and orexin, which are involved in the control of energy balance, may be chemical mediators in the brain that are responsible for the generation and maintenance of hypertension.

In conclusion, in addition to their potent effects on appetite, orexins may interact with the brain system, controlling sympathetic outflow and cardiovascular function and may, therefore, have a broader spectrum of action than previously hypothesized.

Perspectives