Vasodilator Effects of Elcatonin, a Synthetic Eel Calcitonin, on Retinal Blood Vessels in Rats

Abstract

The aim of this study was to examine the effects of elcatonin, a synthetic derivative of eel calcitonin, on rat retinal blood vessels, and to determine how diabetes affects the retinal vascular responses. Ocular fundus images were captured with an original high-resolution digital fundus camera in vivo. The retinal vascular responses were evaluated by measuring the diameter of retinal blood vessels contained in the digital images. Both systemic blood pressure and heart rate were continuously recorded. Elcatonin increased the diameter of retinal blood vessels but decreased mean blood pressure in a dose-dependent manner, whereas it had no significant effect on heart rate. A diminished retinal vasodilator response and significant pressor response to elcatonin were observed in rats injected intravenously with N^G-nitro-L-arginine methyl ester, a nitric oxide (NO) synthase inhibitor. Intravitreal injection of indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ22536, an adenylyl cyclase inhibitor, markedly attenuated the vasodilator effects of elcatonin on retinal blood vessels. The retinal vasodilator responses to elcatonin were unaffected 2 weeks after the induction of diabetes by a combination of streptozotocin treatment and D-glucose feeding. These results suggest that elcatonin dilates rat retinal blood vessels via NO- and COX-dependent mechanisms and that the adenylyl cyclase-adenosine 3′,5′-cyclic monophosphate system plays a major role in the vasodilator mechanisms. The retinal vasodilatory effects of elcatonin seem to be preserved at early stages of diabetes.

Diabetic retinopathy is a leading cause of blindness in industrialized countries and is the most common complication of diabetes. Tight control of blood glucose levels decreases the risk of diabetic retinopathy onset and progression; however, additional effective treatments are still needed. The narrowing of retinal blood vessels and reduction in retinal blood flow are associated with the onset of diabetic retinopathy, and the impairment of retinal circulation contributes to the pathogenesis of the disease.^1–4) Therefore, one strategy for preventing the disease or reducing its severity is to improve the impaired retinal circulation.

Our previous studies demonstrated that adenosine 3′,5′-cyclic monophosphate (cAMP)-elevating agents dilate retinal blood vessels more effectively than peripheral resistance vessels.^5,6) For example, prostaglandin (PG) I₂ and forskolin (an activator of adenylyl cyclase), which elevate intracellular cAMP levels, dilate retinal arterioles at doses much lower than that required to decrease systemic blood pressure.⁵⁾ A cAMP analog decreased systemic blood pressure less than that by an analog of guanosine 3′,5′-cyclic monophosphate (cGMP), though they produced a comparable vasodilation of retinal blood vessels.⁶⁾ These results suggested that cAMP-elevating agents could be effective drugs for improving retinal circulation.

Calcitonin, a polypeptide hormone secreted from parafollicular (C) cells of the mammalian thyroid gland into general circulation, regulates blood Ca²⁺ levels and bone metabolism by acting on osteoclasts.^7,8) Therefore, calcitonin is used clinically to treat hypercalcemia⁹⁾ and osteoporosis.^10,11) Calcitonin also has the ability to relieve pain associated with a variety of disorders, including osteoporosis.^12–16) These actions are mediated by increased cAMP and intracellular Ca²⁺ levels evoked by the stimulation of calcitonin receptors coupled to G proteins.^17–19)

Cutaneous flushing is one of the most common side effects of calcitonin and the effects of elcatonin, a synthetic analog of eel calcitonin,²⁰⁾ might be closely associated with plasma levels of vasoactive intestinal peptide.²¹⁾ Previous studies have shown that calcitonin increased renal blood flow²²⁾ and improved peripheral circulatory disturbance in patients with Raynaud’s syndrome.²³⁾ In addition, elcatonin attenuated noradrenaline-induced contractions of plantar arteries isolated from rats with chronic constriction injury of the sciatic nerve.²⁴⁾ Despite its vasodilator effect on the peripheral circulatory system, systemic administration of calcitonin had no significant effect on systemic blood pressure.^25–27) Thus, the vasodilator action of calcitonin appears to vary between vascular beds. Retinal blood vessels have different characteristics compared to blood vessels in other peripheral circulatory beds.^28,29) Therefore, we hypothesized that calcitonin shows vasodilator effects on retinal blood vessels.

To test this hypothesis, we examined the effects of elcatonin on retinal blood vessels in rats and determined the contribution of nitric oxide (NO)- and cyclooxygenase (COX)-dependent mechanisms to the elcatonin-induced responses. In addition, we determined how the retinal vascular responses to elcatonin were affected in an experimental model of diabetes induced by a combination of streptozotocin (STZ) injection and D-glucose feeding.²⁸⁾

MATERIALS AND METHODS

Reagents

The following reagents were used: fluorescein sodium salt, indomethacin, methoxamine hydrochloride, N^G-nitro-L-arginine methyl ester (L-NAME), SQ22536 (Sigma-Aldrich, St. Louis, MO, U.S.A.), pontamine sky blue 6B (Tokyo-Kasei Kogyo, Tokyo, Japan), hydroxyethylcellulose (Scopisol 15^®, Senju Pharmaceutical, Osaka, Japan), pentobarbital sodium, STZ, tetrodotoxin (TTX) (Nacalai Tesque, Kyoto, Japan), and elcatonin (Asahi Kasei Pharma Corporation, Tokyo, Japan).

Elcatonin was dissolved in 0.01 mM sodium acetate buffer (pH 5.6) containing 0.02% bovine serum albumin and 0.9% sodium chloride and further diluted in the same buffer (4 or 40 U/mL). L-NAME was dissolved in saline (30 mg/mL). Indomethacin and SQ22536 were dissolved in dimethyl sulfoxide (DMSO) (10 and 100 mM for indomethacin and SQ22536, respectively) and further diluted in saline (1 and 10 mM for indomethacin and SQ22536, respectively).

Animals

Forty-four male Wistar rats (6–9 weeks old) were maintained in a room with constant temperature (22±2°C), humidity (55±5%), and a 12-h light/dark cycle and were allowed free access to standard rat chow and tap water.

All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research, and the Regulations for the Care and Use of Laboratory Animals in Kitasato University adopted by the Institutional Animal Care and Use Committee for Kitasato University.

Experimental Procedures

Surgical procedures were performed under general anesthesia with pentobarbital sodium (50 mg/kg, intraperitoneally (i.p.)) following the procedure described in our previous reports.^5,30) Digitized mean arterial pressure (MAP) and heart rate (HR) were stored on the hard disk of a personal computer. It was necessary to prevent eye movements for the capture of fundus images at the same angle throughout the experiment. Therefore, the anesthetized rats were treated with TTX (50 µg/kg, intravenously (i.v.)) under artificial ventilation with room air (stroke volume, 10 mL/kg; frequency, 80 strokes/min) using a rodent respirator (SN-480-7, Shinano, Tokyo, Japan). To compensate for the decrease in blood pressure induced by TTX, methoxamine was continuously infused using a syringe pump (Model 1140-001, Harvard Apparatus, South Natick, MA, U.S.A.). The dose of methoxamine was ca. 40 µg/kg/min (ca. 5.5 µg/kg/min for L-NAME-treated rats).

Experimental Protocols

Protocol 1: To investigate the role of NO in elcatonin-induced responses, we examined the effects of the NO synthase inhibitor L-NAME on elcatonin-induced changes in the diameter of retinal blood vessels, MAP, and HR. L-NAME (30 mg/kg, i.v.) was administered, and methoxamine infusion was started 45 min later. After hemodynamic parameters had reached a stable level (ca. 15 min later), elcatonin (1–30 U/kg/min) was injected into the femoral vein using a syringe pump (Model 1140-001, Harvard Apparatus).

Protocol 2: Our previous findings suggest that NO dilates rat retinal blood vessels through the COX-1/PG/cAMP-mediated signaling pathway^6,31) (Fig. 1). Therefore, we examined the role of the PG/cAMP system in the elcatonin-induced vasodilation of retinal blood vessels. The COX inhibitor indomethacin (1 mM), the adenylyl cyclase inhibitor SQ22536 (10 mM), or the vehicle (10% DMSO) in a total volume of 10 µL was injected into the vitreous cavity of the left eye prior to the surgical procedures and the TTX treatment described above. Intravitreal injection was performed as previously reported.^6,32,33) About 90 min following the intravitreal injection, elcatonin (1–30 U/kg/min, i.v.) was infused using a syringe pump (Model 1140-001, Harvard Apparatus).

Fig. 1. Proposed Mechanisms by Which Nitric Oxide (NO) Dilates Rat Retinal Blood Vessels

NO activates preferentially the cyclooxygenase-1 (COX-1)/prostaglandin (PG)/cAMP-mediated pathway rather than the soluble guanylyl cyclase (sGC)/cGMP-mediated pathway. AC; adenylyl cyclase, eNOS; endothelial NO synthase, PLA₂; phospholipase A₂, EP2; PGE₂ receptor EP2 subtype, PGES; PGE₂ synthase, IP; prostacyclin (PGI₂) receptor, PGIS; PGI₂ synthase.

The timing of administration and the dose of each compound for “Protocol 1” and “Protocol 2” were selected based on results of our previous studies.^6,31)

Protocol 3: To investigate how retinal vasodilator responses to elcatonin are affected by diabetes mellitus, we examined responses to elcatonin in STZ-induced diabetic rats. A single injection of STZ (65 mg/kg, i.v.) dissolved in sodium citrate buffer (pH 4.5) or an equal volume of the buffer was performed. The STZ-treated animals were given drinking water containing 5% D-glucose following treatment with STZ to shorten the term in the development of retinal vascular dysfunction.²⁸⁾ The experiments were performed 2 weeks after the injection. Plasma glucose was determined with a commercially available enzyme kit (Glucose Test Wako, Wako Pure Chemical Industries, Ltd., Osaka, Japan). In this model, retinal vasodilator responses to acetylcholine and β₂-adrenoceptor agonists, but not to forskolin and NO donors, were markedly diminished 2 weeks following the onset of diabetes.^28,29,33)

Fundus Photography and Measurement of Retinal Blood Vessel Diameter

Fundus photography was performed as described in our previous studies.^5,31) Briefly, sodium fluorescein (10%, 0.8 mL/kg) and pontamine sky blue 6B (5%, 0.8 mL/kg) solutions were injected into the right femoral vein to enhance blood vessel contrast. To prevent drying of the eye, hydroxyethylcellulose (Scopisol 15^®) was administered to the cornea. Fundus images were captured before and during i.v. infusion of elcatonin, using a digital camera (EOS7D, Canon, Tokyo, Japan) equipped with a bore scope-type objective lens for small animals (Model 01; Scalar, Tokyo, Japan). Fundus images (5184×3456 pixels, pixel size=1 µm) were stored on the hard disk of a laboratory computer system. A region of the fundus image containing an arteriole or a venule was selected (140×280 pixels, 140×280 µm), and the vessel diameter in the same region was measured throughout the experiment. The retinal vascular response was evaluated by measuring changes in the diameter. The diameter of the retinal blood vessel was expressed as a percentage of the baseline value, just before the infusion of elcatonin.

Data Analyses

All values are presented as mean±standard error (S.E.) To compare baseline values between 2 groups and more than 2 groups, we used unpaired t-test and Tukey’s test, respectively. When comparing the responses between groups, two-way ANOVA was used (PRISM6, GraphPad Software, San Diego, CA, U.S.A.). A p value <0.05 was considered statistically significant.

RESULTS

Baseline values of retinal blood vessel diameter, MAP, and HR are summarized in Table 1. There were no significant differences in the baseline values between experimental groups.

Table 1. Baseline Values of Retinal Arteriolar Diameter (AD), Retinal Venular Diameter (VD) Mean Arterial Pressure (MAP) and Heart Rate (HR) in Rats

Groups	AD (µm)	VD (µm)	MAP (mmHg)	HR (beats/min)
Experiment 1
Saline (n=4)	33.8±0.8	50.9±2.5	115±1	326±12
L-NAME (n=5)	36.1±1.0	48.8±2.8	113±0	316±8
Experiment 2-1
Vehicle (10% DMSO, n=4)	34.3±2.0	50.6±1.9	115±2	321±6
Indomethacin (n=5)	35.0±1.3	50.8±1.8	114±2	337±13
Experiment 2-2
Vehicle (10% DMSO, n=7)	55.4±2.3	65.7±2.9	115±1	352±16
SQ22536 (n=7)	52.2±3.2	59.3±0.8	117±1	356±10
Experiment 3
Non-diabetes (n=6)	45.1±4.1	73.0±9.2	114±1	335±7
Diabetes (n=6)	53.7±5.3	76.1±10.5	112±1	329±7

Values are means±S.E. These values were measured just before starting the infusion of elcatonin.

Effects of L-NAME on Elcatonin-Induced Responses

Elcatonin (1–30 U/kg/min, i.v.) increased the diameter of retinal blood vessels but decreased MAP in a dose-dependent manner (Figs. 2A–C), whereas it had no significant effect on HR (Fig. 2D). The vasodilator effects of elcatonin on retinal blood vessels were significantly attenuated by L-NAME (e.g., vasodilation of retinal arterioles induced by 30 U/kg/min elcatonin; vehicle, 23.9±3.3% vs. L-NAME, 7.0±1.0%) (Figs. 2A, B). In rats treated with L-NAME, elcatonin produced a significant pressor response (e.g., increases in MAP induced by 30 U/kg/min elcatonin were 14.7±5.7%) (Fig. 2C).

Fig. 2. Effects of Intravenous Injection of L-NAME (30 mg/kg) on Changes in Retinal Arteriolar Diameter (AD, A), Retinal Venular Diameter (VD, B), Mean Arterial Pressure (MAP, C) and Heart Rate (HR, D) Induced by Intravenous Infusion of Elcatonin (1–30 U/kg/min)

Data are expressed as percentage of the baseline level (values measured just before starting the infusion). Each point with a vertical bar represents the mean±S.E. from four-five animals. * p<0.05, ^# p<0.05 vs. the corresponding “Time 0” values of the vehicle-treated group, ^§ p<0.05 vs. the corresponding “Time 0” values of the L-NAME-treated group.

Effects of Indomethacin and SQ22536 on Elcatonin-Induced Responses

Intravitreal injection of indomethacin significantly attenuated elcatonin-induced vasodilation of retinal blood vessels (e.g., vasodilation of retinal arterioles induced by 30 U/kg/min elcatonin; vehicle, 26.1±3.0% vs. indomethacin, 3.8±1.1%) (Fig. 3). However, it had no significant effects on MAP and HR responses to elcatonin (data not shown). Similar observations were made with SQ22536 (Fig. 4).

Fig. 3. Effects of Intravitreal Injection of Indomethacin (10 nmol/Eye) on Changes in Retinal Arteriolar Diameter (AD, A) and Retinal Venular Diameter (VD, B) Induced by Intravenous Infusion of Elcatonin (1–30 U/kg/min)

Data are expressed as percentage of the baseline level (values measured just before starting the infusion). Each point with a vertical bar represents the mean±S.E. from four-five animals. * p<0.05, ^# p<0.05 vs. the corresponding “Time 0” values of the vehicle-treated group.

Fig. 4. Effects of Intravitreal Injection of SQ22536 (100 nmol/Eye) on Changes in Retinal Arteriolar Diameter (AD, A) and Retinal Venular Diameter (VD, B) Induced by Intravenous Infusion of Elcatonin (1–30 U/kg/min)

Data are expressed as percentage of the baseline level (values measured just before starting the infusion). Each point with a vertical bar represents the mean±S.E. from seven animals. * p<0.05, ^# p<0.05 vs. the corresponding “Time 0” values of the vehicle-treated group.

Characteristics of Diabetic Rats

On the day of experimentation, plasma glucose levels in diabetic rats were significantly higher than those in non-diabetic rats. In contrast, the body weights of diabetic rats were lower than that of non-diabetic rats (Table 2). Baseline values were adjusted to the same ranges between non-diabetic and diabetic groups by changing the methoxamine infusion rates (Table 1).

Table 2. Plasma Glucose Concentration and Body Weight Measured Just before (Initial) and 2 Weeks (Final) after the Injection

Groups	Plasma glucose (mg/dL)		Body weight (g)
	Initial	Final	Initial	Final
Non-diabetes (n=6)	120±8	110±4	170±5	236±15a)
Diabetes (n=6)	114±7	824±47a,b)	170±6	171±16b)

Values are mean±S.E. The experiments were performed 2 weeks after injection of streptozotocin or the vehicle. a) p<0.05 vs. the corresponding initial value. b) p<0.01 vs. non-diabetes group.

Effects of Diabetes on Elcatonin-Induced Responses

There were no significant differences in retinal vasodilator responses to elcatonin between non-diabetic and diabetic rats (Figs. 5A, B). The effects of elcatonin on MAP and HR were also unaffected by diabetes (Figs. 5C, D).

Fig. 5. Changes in Retinal Arteriolar Diameter (AD, A), Retinal Venular Diameter (VD, B), Mean Arterial Pressure (MAP, C) and Heart Rate (HR, D) Induced by Intravenous Infusion of Elcatonin (1–30 U/kg/min) in Non-diabetic (Non-diabetes) and Diabetic Rats (Diabetes)

Data are expressed as percentage of the baseline level (values measured just before starting the infusion). Each point with a vertical bar represents the mean±S.E. from six animals. ^# p<0.05 vs. the corresponding “Time 0” values of the non-diabetes group, ^§ p<0.05 vs. the corresponding “Time 0” values of the diabetes group.

DISCUSSION

The present study demonstrates that elcatonin increased the diameter of retinal blood vessels in a dose-dependent manner. The elcatonin-induced vasodilation of retinal blood vessels was significantly reduced by L-NAME and indomethacin. In addition, SQ22536, an inhibitor of adenylyl cyclase, almost completely prevented the retinal vasodilator responses. These results suggest that elcatonin dilates rat retinal blood vessels via NO- and COX-dependent mechanisms, and that the adenylyl cyclase-cAMP system plays an important role in the vasodilator mechanisms.

The calcitonin receptor is a member of a subfamily of the G-protein coupled receptor superfamily and couples to adenylyl cyclase and phospholipase C.^17–19) The current study shows that SQ22536 almost completely prevented the elcatonin-induced vasodilation of retinal blood vessels. Thus, the adenylyl cyclase-cAMP system seems to play an important role in the retinal vasodilator response to elcatonin. However, in rat retinal blood vessels, the stimulatory effect of elcatonin on the adenylyl cyclase-cAMP system is likely to be indirectly mediated by NO- and COX-dependent mechanisms, because retinal vasodilator responses to elcatonin were prevented by L-NAME and indomethacin. In most vascular beds, NO dilates blood vessels by activating soluble guanylyl cyclase and elevating intracellular cGMP levels,³⁴⁾ whereas the NO-induced vasodilator response in rat retinal blood vessels is mediated mainly through the COX-1/PG/cAMP-mediated signaling pathway.^6,31) Therefore, the NO-dependent component of the elcatonin-induced response is likely mediated by stimulation of the COX/cAMP pathway, not that of soluble guanylyl cyclase/cGMP pathway. Presently, the mechanism by which elcatonin stimulates the NO-mediated signaling pathway remains unclear.

We have shown that retinal blood vessels have different characteristics compared to blood vessels in other peripheral circulatory beds.^28,29) The present study also provides evidence suggesting that responses evoked by the stimulation of calcitonin receptors may differ between retinal and peripheral vasculatures because elcatonin dilated retinal blood vessels in an NO- and COX-dependent manner, whereas it decreased blood pressure in an NO-dependent manner. Interestingly, in the presence of L-NAME, elcatonin exhibited a significant pressor effect. These results indicate that the contractile action of elcatonin may usually be counteracted by its vasodilatory action, involving NO. Peripheral administration of elcatonin increased plasma renin activity but did not change blood pressure in rats.²⁵⁾ Perhaps, contractile action due to increased plasma renin activity may be counteracted by an intact NO-mediated vasodilatory effect.

To address how diabetes affects the elcatonin-induced vasodilation of retinal blood vessels, we examined the effects of elcatonin in an experimental model of diabetes induced by a combination of STZ injection and D-glucose feeding. In the same model, we found that the vasodilator effects of elcatonin on retinal blood vessels are unaffected after a 2-week duration of diabetes. At the stage of this model, retinal vasodilator responses to acetylcholine and β₂-adrenoceptor agonists, but not to forskolin and NO donors, are diminished.^28,29,33) The NO- and prostaglandin-independent, possibly endothelium-derived hyperpolarizing factor (EDHF)-mediated, component of acetylcholine-induced vasodilation of retinal blood vessels is affected in diabetic rats.³³⁾ Therefore, the vasodilatory effects of elcatonin are unlikely to be mediated by EDHF or β₂-adrenoceptor stimulation. The vasodilatory mechanisms of elcatonin in retinal blood vessels seem to be preserved at the early stage of diabetes.

It is well known that endothelial function in systemic circulation is impaired in diabetic animals.^35–37) Therefore, elcatonin might cause a significant pressor response in diabetic rats as demonstrated in L-NAME-treated rats; however, we did not observe any significant pressor effect of elcatonin in our diabetic model. In the model, depressor responses to acetylcholine are not affected after 2 weeks of diabetes; therefore, endothelial NO-mediated vasodilatory mechanisms in peripheral resistance vessels may be preserved at early stages of diabetes.²⁸⁾ Thus, an intact endothelium-dependent vasodilatory effect may counteract the contractile action of elcatonin.

In summary, the present study provides the first pharmacological evidence indicating that elcatonin dilates retinal blood vessels. Impairment of retinal circulation could contribute to the pathogenesis of diabetic retinopathy.^1–4) Therefore, elcatonin could become a candidate therapeutic for preventing progression of diabetic retinopathy by improving retinal circulation. However, the detailed mechanism of the retinal vasodilator response to elcatonin remains unclear, and calcitonin may stimulate tumor angiogenesis through the calcitonin receptor.³⁸⁾ Further studies are needed to address these points.

Acknowledgments

This study was supported by the Kitasato University Research Grant for Young Researchers (A. M.), JSPS KAKENHI (Grant Numbers: 21590102 K. I., 24790261 A. M. and 24590122 K. I.), and MEXT KAKENHI (Grant Number: 25122712, T. N.).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Feke GT, Buzney SM, Ogasawara H, Fujio N, Goger DG, Spack NP, Gabbay KH. Retinal circulatory abnormalities in type 1 diabetes. Invest. Ophthalmol. Vis. Sci., 35, 2968–2975 (1994).
• 2) Ghirlanda G, Di Leo MA, Caputo S, Cercone S, Greco AV. From functional to microvascular abnormalities in early diabetic retinopathy. Diabetes Metab. Rev., 13, 15–35 (1997).
• 3) Schmetterer L, Wolzt M. Ocular blood flow and associated functional deviations in diabetic retinopathy. Diabetologia, 42, 387–405 (1999).
• 4) De La Cruz JP, González-Correa JA, Guerrero A, de la Cuesta FS. Pharmacological approach to diabetic retinopathy. Diabetes Metab. Res. Rev., 20, 91–113 (2004).
• 5) Mori A, Saito M, Sakamoto K, Narita M, Nakahara T, Ishii K. Stimulation of prostanoid IP and EP₂ receptors dilates retinal arterioles and increases retinal and choroidal blood flow in rats. Eur. J. Pharmacol., 570, 135–141 (2007).
• 6) Ogawa N, Mori A, Hasebe M, Hoshino M, Saito M, Sakamoto K, Nakahara T, Ishii K. Nitric oxide dilates rat retinal blood vessels by cyclooxygenase-dependent mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol., 297, R968–R977 (2009).
• 7) Nicholson GC, Moseley JM, Sexton PM, Mendelsohn FA, Martin TJ. Abundant calcitonin receptors in isolated rat osteoclasts. Biochemical and autoradiographic characterization. J. Clin. Invest., 78, 355–360 (1986).
• 8) Suzuki H, Nakamura I, Takahashi N, Ikuhara T, Matsuzaki K, Isogai Y, Hori M, Suda T. Calcitonin-induced changes in the cytoskeleton are mediated by a signal pathway associated with protein kinase A in osteoclasts. Endocrinology, 137, 4685–4690 (1996).
• 9) Silva OL, Becker KL. Salmon calcitonin in the treatment of hypercalcemia. Arch. Intern. Med., 132, 337–339 (1973).
• 10) Jiménez FE, Albizuri JMA, Alier JMA, Soto JJM, Canales AG. Effectiveness and safety of medium- and long-term elcatonin use in the prevention and treatment of bone mass loss. Curr. Ther. Res., 56, 385–399 (1995).
• 11) Orimo H, Morii H, Inoue T, Yamamoto K, Minaguchi H, Ishii Y, Murota K, Fujimaki E, Watanabe R, Harata S, Honjo H, Fujita T. Effect of elcatonin on involutional osteoporosis. J. Bone Miner. Metab., 14, 73–78 (1996).
• 12) Quatraro A, Minei A, De Rosa N, Giugliano D. Calcitonin in painful diabetic neuropathy. Lancet, 339, 746–747 (1992).
• 13) Pontiroli AE, Pajetta E, Scaglia L, Rubinacci A, Resmini G, Arrigoni M, Pozza G. Analgesic effect of intranasal and intramuscular salmon calcitonin in post-menopausal osteoporosis: a double-blind, double-placebo study. Aging Milano, 6, 459–463 (1994).
• 14) Shibata K, Takeda M, Ito A, Takeda M, Sagai H. Ovariectomy-induced hyperalgesia and antinociceptive effect of elcatonin, a synthetic eel calcitonin. Pharmacol. Biochem. Behav., 60, 371–376 (1998).
• 15) Aoki M, Mori A, Nakahara T, Sakamoto K, Ishii K. Effect of synthetic eel calcitonin, elcatonin, on cold and mechanical allodynia induced by oxaliplatin and paclitaxel in rats. Eur. J. Pharmacol., 696, 62–69 (2012).
• 16) Ito A, Takeda M, Yoshimura T, Komatsu T, Ohno T, Kuriyama H, Matsuda A, Yoshimura M. Anti-hyperalgesic effects of calcitonin on neuropathic pain interacting with its peripheral receptors. Mol. Pain, 8, 42 (2012).
• 17) Lin HY, Harris TL, Flannery MS, Aruffo A, Kaji EH, Gorn A, Kolakowski LF Jr, Lodish HF, Goldring SR. Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science, 254, 1022–1024 (1991).
• 18) Force T, Bonventre JV, Flannery MR, Gorn AH, Yamin M, Goldring SR. A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C. Am. J. Physiol., 262, F1110–F1115 (1992).
• 19) Masi L, Brandi ML. Calcitonin and calcitonin receptors. Clin. Cases Miner. Bone Metab., 4, 117–122 (2007).
• 20) Morikawa T, Munekata E, Sakakibara S, Noda T, Otani M. Synthesis of eel-calcitonin and (asu1,7)-eel-calcitonin: contribution of the disulfide bond to the hormonal activity. Experientia, 32, 1104–1106 (1976).
• 21) Takeyama M, Ikawa K, Nagano T, Mori K. Elcatonin raises levels of vasoactive intestinal peptide in human plasma. J. Pharm. Pharmacol., 48, 657–659 (1996).
• 22) Edwards IR, Smith AJ. Porcine calcitonin as a renal vasodilator in man. Clin. Endocrinol., 1, 337–344 (1972).
• 23) Franchi F, Chiarini P, Matassi L, Strazzulla G, Scardi A, Righi D, Nuzzaci G. Effects of calcitonin on limb blood vessels in human obstructive arterial disease. Int. J. Clin. Pharmacol. Res., 3, 115–128 (1983).
• 24) Yoshimura T, Ito A, Saito SY, Takeda M, Kuriyama H, Ishikawa T. Calcitonin ameliorates enhanced arterial contractility after chronic constriction injury of the sciatic nerve in rats. Fundam. Clin. Pharmacol., 26, 315–321 (2012).
• 25) Clementi G, Rapisarda E, Fiore CE, Prato A, Amico-Roxas M, Millia C, Bernardini R, Maugeri S, Scapagnini U. Effects of salmon calcitonin on plasma renin activity and systolic blood pressure in the rat. Neurosci. Lett., 66, 351–355 (1986).
• 26) Wegener LL, McCarron DA. Acute intravenous calcitonin: failure to modify systemic blood pressure. Peptides, 9, 1191–1193 (1988).
• 27) Fujimoto S, Fujimoto SK. Elcatonin-mediated contractile and relaxant responses in SHR femoral artery. Acta Pharmacol. Sin., 22, 595–602 (2001).
• 28) Mori A, Saigo O, Hanada M, Nakahara T, Ishii K. Hyperglycemia accelerates impairment of vasodilator responses to acetylcholine of retinal blood vessels in rats. J. Pharmacol. Sci., 110, 160–168 (2009).
• 29) Mori A, Miwa T, Sakamoto K, Nakahara T, Ishii K. Pharmacological evidence for the presence of functional β₃-adrenoceptors in rat retinal blood vessels. Naunyn Schmiedebergs Arch. Pharmacol., 382, 119–126 (2010).
• 30) Kaneko Y, Saito M, Mori A, Sakamoto K, Nakahara T, Ishii K. Vasodilator effects of adrenomedullin on retinal arterioles in streptozotocin-induced diabetic rats. J. Ocul. Pharmacol. Ther., 22, 317–322 (2006).
• 31) Ogawa N, Saito M, Mori A, Sakamoto K, Kametaka S, Nakahara T, Ishii K. Vasodilator effect of nicorandil on retinal blood vessels in rats. Naunyn Schmiedebergs Arch. Pharmacol., 375, 323–328 (2007).
• 32) Mori A, Suzuki S, Sakamoto K, Nakahara T, Ishii K. Role of calcium-activated potassium channels in acetylcholine-induced vasodilation of rat retinal arterioles in vivo. Naunyn Schmiedebergs Arch. Pharmacol., 383, 27–34 (2011).
• 33) Mori A, Suzuki S, Sakamoto K, Nakahara T, Ishii K. Vasodilation of retinal arterioles induced by activation of BK_Ca channels is attenuated in diabetic rats. Eur. J. Pharmacol., 669, 94–99 (2011).
• 34) Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev., 43, 109–142 (1991).
• 35) Oyama Y, Kawasaki H, Hattori Y, Kanno M. Attenuation of endothelium-dependent relaxation in aorta from diabetic rats. Eur. J. Pharmacol., 132, 75–78 (1986).
• 36) Taylor PD, McCarthy AL, Thomas CR, Poston L. Endothelium-dependent relaxation and noradrenaline sensitivity in mesenteric resistance arteries of streptozotocin-induced diabetic rats. Br. J. Pharmacol., 107, 393–399 (1992).
• 37) De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM. Endothelial dysfunction in diabetes. Br. J. Pharmacol., 130, 963–974 (2000).
• 38) Chigurupati S, Kulkarni T, Thomas S, Shah G. Calcitonin stimulates multiple stages of angiogenesis by directly acting on endothelial cells. Cancer Res., 65, 8519–8529 (2005).