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. 1999 Dec 1;521(Pt 2):507–516. doi: 10.1111/j.1469-7793.1999.00507.x

Mechanisms underlying spontaneous rhythmical contractions in irideal arterioles of the rat

Caryl E Hill 1, Joyce Eade 1, Shaun L Sandow 1
PMCID: PMC2269675  PMID: 10581319

Abstract

  1. Mechanisms underlying spontaneous rhythmical contractions have been studied in irideal arterioles of the rat using video microscopy and electrophysiology.

  2. Rhythmical contractions (4 min−1) were more common during the second and third postnatal weeks and were always preceded by large, slow depolarizations (5-40 mV).

  3. Spontaneous contractions were unaffected by tetrodotoxin (1 μm), neurotransmitter receptor antagonists, the sympathetic neurone blocker, guanethidine (5 μm) or sensory neurotoxin, capsaicin (1 μm).

  4. Stimulation of sensory nerves inhibited spontaneous activity and this was not prevented by L-NAME (10 μm).

  5. L-NAME (10 μm) caused an increase in frequency of spontaneous contractions, while forskolin (30 nm), in the presence of L-NAME, abolished spontaneous, but not nerve-mediated, contractions.

  6. Spontaneous activity was not affected by felodipine (1 nm) or nifedipine (1 μm), but was abolished by cadmium chloride (1 μm) or superfusion with calcium-free solution.

  7. Caffeine (1 mm), thapsigargin (2 μm) and cyclopiazonic acid (3 μm), but not ryanodine (3 μm), abolished spontaneous and nerve-mediated contractions. After preincubation in L-NAME (10 μm), cyclopiazonic acid abolished spontaneous contractions only.

  8. Spontaneous depolarizations and contractions were abolished by 18α-glycyrrhetinic acid (20 μm).

  9. Results suggest that spontaneous rhythmical contractions are myogenic and result from the cyclical release of calcium from intracellular stores, without a contribution from voltage-dependent calcium channels. Intercellular coupling through gap junctions appears to be essential for co-ordination of these events which could be modulated by nitric oxide and increases in cAMP. The possibility that different intracellular stores underly spontaneous and nerve-mediated contractions is discussed.

A number of smooth muscle tissues have been observed to undergo spontaneous, rhythmical contractions. These include the gastrointestinal tract (Tomita, 1981), urinary tract (Zhang & Lang, 1994) and urethra (Hashitani et al. 1996), lymphatics (van Helden, 1993) and blood vessels. Rhythmical contractions, or vasomotion, have been observed in vivo in small arterioles in the cerebral circulation (Hundley et al. 1988; Morita-Tsuzuki et al. 1992), in mucosal surfaces (Bouskela & Grampp, 1992) and in skeletal muscle (Bertuglia et al. 1994), and have been proposed to play a role in vascular resistance and blood flow (Gratton et al. 1998). In vitro, rhythmical contractions in blood vessels have been described in isolated pressurized arterioles (Duling et al. 1981), following treatment with agonists (Gustafsson, 1993) or increases in the extracellular concentration of potassium ions (Katusic et al. 1988; Gokina et al. 1996) or when some vascular muscle strips are set up under isometric conditions (Lee et al. 1994; Stork & Cocks, 1994; Gokina et al. 1996). Spontaneous contractions have also been reported to occur in some elastic arteries in vivo and in vitro after exposure to agonist (Hayashida et al. 1986; Chemtob et al. 1992; Porret et al. 1995; Eddinger & Ratz, 1997).

In the rat portal vein, mesenteric arteries, thoracic aorta and carotid artery, human pial and epicardial coronary arteries, rabbit femoral, mesenteric and ear arteries, rhythmical contractions were completely abolished following the application of antagonists against voltage-dependent calcium channels (Hayashida et al. 1986; Chemtob et al. 1992; Gustafsson, 1993; Omote & Mizusawa, 1993; Omote et al. 1993; Stork & Cocks, 1994; Omote & Mizusawa, 1995; Gokina et al. 1996; Miwa et al. 1997; Okumura et al. 1997). In small arteries of the rat mesentery, however, these rhythmical contractions were also abolished with substances which interfere with the release of calcium from intracellular stores (Gustafsson & Nilsson, 1993), suggesting that an interplay between extracellular and intracellular calcium may be responsible for these spontaneous contractions which appear following treatment with agonist.

In guinea-pig lymphatics and rabbit urethra, spontaneous contractions have been observed to be preceded by large, rhythmical depolarizations (van Helden, 1993; Hashitani et al. 1996). While the spontaneous contractions were abolished by treatment with voltage-dependent calcium-channel antagonists, the spontaneous depolarizations have been shown to result from the opening of chloride channels in response to the release of intracellular calcium (van Helden, 1993; Hashitani et al. 1996; von der Weid et al. 1996). Thus, the spontaneous contractions in these tissues resulted from the influx of calcium through voltage-dependent calcium channels secondary to the activation of calcium-dependent channels by release of intracellularly stored calcium.

In arterioles of the adult rat iris, constriction in response to sympathetic nerve stimulation has been reported to result from the activation of α1B-adrenoceptors and the release of intracellular calcium (Gould & Hill, 1994, 1996). The released calcium also activates a chloride channel in the cell membrane leading to a large depolarization which precedes the contraction (Gould & Hill, 1996). These small vessels sometimes exhibit spontaneous contractions and when recorded, these have been shown to be preceded by spontaneous depolarizations which resemble those recorded following nerve stimulation (Gould & Hill, 1996). In the present paper, the occurrence and mechanism underlying these spontaneous contractions have been investigated. We report that spontaneous contractions in irideal arterioles are common during early postnatal development. The contractions are myogenic, result from the release of intracellular calcium and are independent of the activity of voltage-dependent ion channels. Some of the results have been previously presented in abstract form (Hill et al. 1998).

METHODS

Tissue preparation

Wistar rats aged 7-28 days postnatal of either sex, were killed humanely with an overdose of ether anaesthetic, followed by cervical dislocation. All experiments were performed in accordance to the guidelines of the Animal Experimentation Ethics Committee of the Australian National University. The eyes were removed and the iris and cornea isolated. The iris was cut in half and pinned along the corneal and sphincter edges in a recording chamber, which was continually perfused at 3 ml min−1 with Krebs solution of the following composition (mm): NaCl, 119.8; KCl, 5.0; NaHCO3, 25; NaH2PO4, 1.0; CaCl2, 2.5; MgCl2, 2.0; glucose, 22.0. The Krebs solution was gassed with 95 % O2-5 % CO2 and maintained at 33-34°C.

Initial experiments to determine the incidence of spontaneous contractions during development were carried out on arterioles from rats aged 7-28 days. Experiments to investigate the mechanism underlying the spontaneous contractions were conducted on rats aged between 16 and 21 days. For these latter experiments, controls were performed to determine the time over which spontaneous contractions were constant in frequency and amplitude. In all cases, results were obtained from a minimum of four preparations, each from a different rat.

All preparations were equilibrated for 20 min. Nerves were stimulated at 10 Hz for 1 s (pulses of 0.1 ms, 100 mA) with a pair of platinum electrodes placed on either side of the preparation. This was done at the beginning of an experiment and then later after drugs had been present for at least 10 min. Drugs were superfused for 20 min before returning to control Krebs solution. Nerves were stimulated again after 10 min in control Krebs solution. In the case of the irreversible α1-adrenoceptor antagonist, benextramine, the drug was present for 10 min until the response to nerve stimulation was abolished, and then the preparation was superfused with control Krebs solution to prevent non-specific side effects. Hyoscine hydrochloride (1 μm) was present in all experiments to prevent the effect of stimulation of cholinergic nerves. Capsaicin (1 μm) was present in some experiments to prevent the action of sensory nerves. Spontaneous and nerve-mediated contractions were recorded with a video camera and computer program (Diamtrak; Neild, 1989).

Intracellular recordings were made from the same sites where vessel diameter was monitored using conventional techniques with fine borosilicate glass microelectrodes, filled with 0.5 M KCl, and having resistances of 120-220 MΩ (Flaming Brown micropipette puller, Sutter Instrument Co). Membrane potential was measured with an Axoclamp 2B (Axon Instruments). All membrane potential records were low-pass filtered, cut-off frequency 1 kHz. Simultaneous changes in membrane potential and changes in vessel diameter in the region where the cell was impaled were acquired with sample rates of greater than 100 Hz and stored on computer disk for analysis.

Solutions and chemicals

The following drugs were purchased commercially: benextramine tetrachloride, (-)-hyoscine (scopolamine) hydrochloride, guanethidine sulphate, tetrodotoxin, cyclopiazonic acid, forskolin, nifedipine, 18α-glycyrrhetinic acid (Sigma); caffeine (Ajax Chemicals, Sydney, Australia); L-NAME, D-NAME hydrochloride (Sapphire Bioscience Pty Ltd, Alexandria, Australia); capsaicin (Fluka Chemie, Switzerland); thapsigargin (Alamone Labs, Jerusalem, Israel); ryanodine (Biomol., Plymouth Meeting, PA, USA); felodipine (Astra Hassle AB, Sweden); pyridoxal phosphate-6-azophenyl-2′,4′ disulphonic acid tetrasodium (PPADS, Research Biochemical Inc., USA). The neuropeptide Y (NPY) Y1 antagonist, 1229U91, was kindly supplied by Professor J. Angus and Dr R. Murphy, University of Melbourne, Australia. Stock solutions (1000-fold) of felodipine and nifedipine were dissolved in absolute alcohol; thapsigargin, 18α-glycyrrhetinic acid and cyclopiazonic acid were dissolved in DMSO and tetrodotoxin in dilute acetate buffer. Control experiments using Krebs solution containing appropriate dilutions of absolute alcohol and DMSO showed there were no effects of the diluent on either contractions or depolarizations. Caffeine was dissolved directly into Krebs solution by stirring and gentle heating. All other drugs were made up as ×1000 stock solutions in distilled water and diluted into Krebs solution. Solutions of light-sensitive drugs were protected from light using aluminium foil and illuminating preparations with only long wavelength (> 610 nm) light. Krebs solution, which was nominated as calcium free, was made by omission of CaCl2.

Statistics

Statistical significance was tested using Student's unpaired t tests or for group values, by one-way ANOVA followed by pairwise t tests with Bonferroni correction for multiple group comparisons. Data were tested for significance using linear regression analysis (GraphPad Prism). A P value of < 0.05 was taken as significant.

RESULTS

Developmental appearance of spontaneous contractions

Arterioles from rats aged 7-28 days were sometimes observed to undergo spontaneous rhythmical contractions. The percentage of preparations exhibiting these contractile events increased during the second postnatal week to reach a maximum at day 14 and decreased after this age (Fig. 1A). The amplitude of spontaneous contractions also increased during development to a maximum at 21 days postnatal (Fig. 1B), while the frequency of contractions varied little during development from a value of about 4 min−1 (Fig. 1C).

Figure 1. Changes in spontaneous contractions during development.

Figure 1

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A, the percentage of preparations containing a spontaneously contracting arteriole is plotted against the postnatal age of the rats. B, the mean amplitude of these spontaneous contractions and C, the frequency of the spontaneous contractions are plotted against postnatal age. At each age, a minimum of 4 rats were used.

When intracellular recordings were made from rats aged 7, 10, 14 and 21 days postnatal, it was found that spontaneous, rhythmical contractions were always preceded by spontaneous, rhythmical depolarizations (Fig. 2). On the other hand, small, spontaneous depolarizations were sometimes observed in preparations which failed to show contractions (Fig. 2A). Spontaneous depolarizations showed variability in shape, some having a smooth form and others showing a spike-like potential on top (Figs 2, 4 and 7). Both types of potential were, however, associated with contractions (Fig. 2A and D) and the onset of the contraction was commonly observed before the peak potential. The amplitude of the depolarizations was positively correlated with the amplitude of contractions (Fig. 3A, linear regression analysis, y-intercept, 13.6 ± 2.0 mV), while there was no correlation between the rise time of the depolarizations (10-90 % peak amplitude) and the amplitude of the contractions (Fig. 3B, linear regression analysis). Rise times appeared to be relatively constant, irrespective of the shape of the potential or its amplitude (Fig. 3).

Figure 2. Spontaneous depolarizations and contractions of arterioles from rats aged 7, 10, 14 and 21 days of age.

Figure 2

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At 7 days of age, spontaneous depolarizations were rarely accompanied by contractions while during the second and third postnatal weeks, spontaneous depolarizations regularly preceded spontaneous contractions. Resting vessel diameters were 32.6, 27.3, 26.0 and 23.5 μm and peak negative membrane potentials were -65, -61, -69 and -65 mV for the arterioles from 7, 10, 14 and 21 days postnatal, respectively.

Figure 4. Sensory nerves and spontaneous contractions.

Figure 4

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The effect of sensory nerves was tested with the neurotoxin, capsaicin (1 μm). In A, in the absence of capsaicin, stimulation of the perivascular nerves (arrow; 10 Hz for 1 s) produces a vasoconstriction and an inhibition of the spontaneous contractions. In B, in the presence of capsaicin, there is no inhibition of spontaneous activity after nerve stimulation. The resting vessel diameter was 27.9 μm in A and 28.3 μm in B for arterioles from 17- and 21-day-old rats, respectively.

Figure 7. Effect of intracellular calcium on spontaneous contractions.

Figure 7

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Spontaneous contractions were abolished following the addition of caffeine (1 mm; A), thapsigargin (2 μm; B) and cyclopiazonic acid (CPA, 3 μm; C) but not by the addition of ryanodine (3 μm; D). At the arrow, the preparations were stimulated transmurally (10 Hz, 1 s). Nerve-mediated contractions were also abolished with the addition of caffeine, thapsigargin and cyclopiazonic acid, but not ryanodine. Addition of L-NAME (10 μm) to iris arterioles to inhibit nitric oxide production did not prevent the spontaneous contractions (E). The subsequent addition of cyclopiazonic acid (CPA, 3 μm), in the continued presence of L-NAME, abolished the spontaneous contractions but had no effect on the nerve-mediated contraction (E). The preparations illustrated were from 21-day-old (A, C, D, E) and 17-day-old (B) rats and had resting vessel diameters of 36.3, 25.5, 36, 36.6 and 26.8 μm, respectively. Addition of caffeine, thapsigargin and cyclopiazonic acid produced a vasodilatation (A, B, C and E) while there was no change in resting vessel diameter in ryanodine (D).

Figure 3. Analysis of spontaneous depolarizations and contractions.

Figure 3

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The amplitude of the spontaneous depolarizations showed a significant correlation with the amplitude of the spontaneous contractions (A; linear regression analysis), while the rise time of the spontaneous depolarizations (B; 10-90 % peak) failed to show any significant correlation with the amplitude of the spontaneous contractions.

No significant changes were found in peak negative membrane potentials of arterioles during development (-63.0 ± 2.2 mV, n = 4 at 7 days; -58.1 ± 3.8 mV, n = 7 at 10 days; -63.1 ± 2.1 mV, n = 12 at 14 days and -64.0 ± 2.7 mV, n = 8 at 21 days; P > 0.05, one-way ANOVA). Furthermore, peak negative membrane potentials of arterioles showing spontaneous depolarizations and contractions did not vary significantly from those of arterioles showing spontaneous depolarizations but no contractions, nor from those of arterioles not showing any spontaneous activity (-64.7 ± 1.0 mV, n = 21; -65.7 ± 1.0 mV, n = 10; -66.6 ± 1.3 mV, n = 11, respectively, P > 0.05, one-way ANOVA).

Time controls

Since spontaneous contractions were most frequently seen during the third postnatal week, experiments to investigate the mechanism underlying the spontaneous contractions were conducted on rats aged between 16 and 21 days. Only preparations which showed regular, rhythmical contractions were used. Control experiments, designed to determine the time over which spontaneous contractions were constant in frequency and amplitude, demonstrated that arterioles showed a progressive development of tone with time in vitro as revealed by a decrease in diameter and that spontaneous, rhythmical contractions regularly appeared after 15-20 min incubation. These contractions were always consistent in amplitude and frequency over the first hour in vitro, frequently lasting for up to one and a half hours. After this time, spontaneous contractions were irregular or absent, although consistent responses to nerve stimulation could be obtained for much longer periods (Gould & Hill, 1994). Consequently, experiments were commenced after 20 min preincubation and concluded within an hour. This allowed time for exposure to one drug per preparation with a return to control solution if the effects of the drug were reversible. The mean diameter of the arterioles studied, when measured at the 20 min time point, was 26.9 ± 0.7 μm (n = 34).

Myogenic origin of spontaneous contractions

In order to determine whether spontaneous contractions could result from the spontaneous release of neurotransmitter, antagonists to receptors activated by the co-transmitters, noradrenaline, NPY and ATP which are released from sympathetic nerves, were tested. The α1-adrenoceptor antagonist, benextramine (10 μm) failed to prevent the spontaneous rhythmical contractions, as did the NPY Y1 antagonist, 1229U91 (300 nm) and the purinergic, P2X1 receptor antagonist, PPADS (10 μm).

The sympathetic neurone blocker, guanethidine (5 μm) also failed to inhibit the spontaneous contractions, supporting the lack of involvement of sympathetic nervous activity. Finally, the sodium channel antagonist, tetrodotoxin (1 μm) had no effect on the spontaneous contractions.

The effect of sensory nerves was investigated using the sensory neurotoxin, capsaicin (1 μm). Spontaneous, rhythmical contractions were seen in preparations in either the absence or presence of capsaicin (Fig. 4). Neither the amplitude nor the frequency was different under these two conditions. However, in the absence of capsaicin, there was a brief inhibition of spontaneous activity immediately following a nerve stimulation and this was absent in the presence of capsaicin (Fig. 4A and B). The inhibitory effect of sensory nerves did not appear to be due to the release of nitric oxide since it persisted in the presence of the nitric oxide synthase inhibitor, L-NAME (10 μm; Fig. 5B).

Figure 5. Effect of cAMP on spontaneous and nerve-mediated depolarizations.

Figure 5

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Spontaneous depolarizations are shown in control solution (A) and in the presence of L-NAME (10 μm) to inhibit nitric oxide production (B), but not following the further addition of forskolin (30 nm; C). Stimulation of perivascular nerves (arrow applies to all three traces, 10 Hz for 1 s) produced a large depolarization under all three conditions. Peak negative membrane potential of the arteriole was -68 mV. Resting vessel diameter was 22.3 μm in A, 19.7 μm in B and 20.6 μm in C.

The addition of L-NAME produced a significant increase in the frequency of spontaneous depolarizations and contractions (Fig. 5B; 134 ± 4.3 %, compared with control, 100 ± 3.1 %, n = 10, P < 0.05). There was no change in the frequency of contractions in the presence of D-NAME (10 μm; 102 ± 5.2 %, compared with control, 100 ± 7.2 %, n = 5, P > 0.05). Furthermore, the addition of L-NAME had no significant effect on the peak negative membrane potential of arterioles (-67.6 ± 1.4 mV, n = 14, compared with control, -65.5 ± 0.7 mV, n = 41, P > 0.05). These results suggest that endogenous release of nitric oxide produces an inhibitory effect on the frequency of spontaneous contractions.

Incubation in the adenylate cyclase activator, forskolin (30 nm), produced a rapid and complete abolition of the spontaneous depolarizations and contractions, along with abolition of the nerve-mediated events. The inhibition of spontaneous activity by forskolin was still seen after preincubation in L-NAME (10 μm; Fig. 5C), to prevent any secondary effects due to the release of nitric oxide (Hill & Gould, 1997). Under these conditions, however, the nerve-mediated depolarization and contraction were no longer abolished (Fig. 5C).

Role of exogenous calcium

Due to the inhibitory effect of sensory nerves on spontaneous contractions illustrated above, the experiments in this section were performed in the presence of capsaicin. Felodipine (1 nm), the antagonist of voltage-dependent calcium channels, failed to affect the spontaneous contractions (Fig. 6A), as did nifedipine (1 μm). On the other hand, the non-selective calcium channel antagonist, cadmium chloride (1 μm) abolished both the rhythmical spontaneous contractions (Fig. 6B) and the nerve-mediated contractions and produced a vasodilatation. On washout, the nerve-mediated responses recovered as did the spontaneous contractions although they were often not as regular as they were initially. Superfusion with calcium-free Krebs solution led to a loss of spontaneous (Fig. 6C) and nerve-mediated contractions in about 4 min. This occurred in the absence of a vasodilatation and loss of tone. On washout, the vessels showed a small dilatation and spontaneous contractions returned. Nerve-mediated contractions were also rapidly restored.

Figure 6. Effect of extracellular calcium on spontaneous contractions.

Figure 6

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Spontaneous contractions were not affected by the L-type calcium channel antagonist, felodipine (1 nm; A), but were abolished with the non-specific calcium channel antagonist, cadmium chloride (1 μm; B) or the removal of calcium from the superfusion solution (C). In A, the arteriole had a resting vessel diameter of 23 μm and was from a 17-day-old rat; in B, the resting vessel diameter was 19 μm and the arteriole was from a 17-day-old rat; and in C, the resting vessel diameter was 30 μm and the arteriole was from a 20-day-old rat.

Role of intracellular calcium

The role of intracellular calcium stores was tested using the calcium mobilizers, caffeine (1 mm) and ryanodine (3 μm), and the calcium-ATPase inhibitors, thapsigargin (2 μm) and cyclopiazonic acid (3 μm). Caffeine, thapsigargin and cyclopiazonic acid all rapidly abolished the spontaneous and nerve-mediated contractions and produced a vasodilatation (Fig. 7A-C), while ryanodine had no effect on either the spontaneous or nerve-mediated contractions, or on resting vessel tone (Fig. 7D). When the effects of cyclopiazonic acid on the release of nitric oxide from the endothelium were prevented by preincubation in L-NAME (10 μm), the spontaneous contractions were abolished but the nerve-mediated contractions were unaffected (Fig. 7E). The abolition of spontaneous contractions was accompanied by a vasodilatation.

Role of gap junctions

The role of gap junctions in the spontaneous contractions was tested with the putative gap junction uncoupling agent, 18α-glycyrrhetinic acid (20 μm). Spontaneous contractions and depolarizations were rapidly abolished within 2-5 min of the addition of 18α-glycyrrhetinic acid and this was accompanied by a vasodilatation (Fig. 8). There was no significant effect of 18α-glycyrrhetinic acid on the peak negative membrane potential of the arterioles (-68.7 ± 1.5 mV, n = 7, compared with control, -65.5 ± 0.7 mV, P > 0.05).

Figure 8. Effect of 18α-glycyrrhetinic acid on spontaneous activity of iris arterioles.

Figure 8

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Spontaneous contractions and depolarizations were rapidly abolished after the addition of 18α-glycyrrhetinic acid (20 μm, +4 min) and the vessel dilated. The resting vessel diameter was 31 μm in control Krebs solution and 35.5 μm in glycyrrhetinic acid. The peak negative membrane potential in glycyrrhetinic acid was -68 mV and the preparation was from a 14-day-old rat.

DISCUSSION

Small arterioles of the rat iris undergo spontaneous rhythmical contractions or vasomotion when placed in vitro and superfused with Krebs solution. The appearance, form and time of cessation of these contractions was very similar to those previously observed in isolated pressurized arterioles from several sources (Duling et al. 1981). Vasomotion in irideal arterioles was most frequently seen in preparations taken from animals aged between 1 and 3 weeks postnatal. It is interesting that the peak of spontaneous activity at 14 days postnatal coincided with eye opening in the rat and with the demonstration of spontaneous contractile movements in the iris dilator muscle itself (Hill et al. 1991). Spontaneous rhythmical contractions of the arterioles may assist in increasing the perfusion of the iris and hence the supply of nutrients to the muscles during this active period. A similar developmental appearance of spontaneous activity has been reported in the carotid artery of young, but not adult, rats exposed to α-adrenoceptor agonists, although this activity peaked at 25 days postnatal (Eddinger & Ratz, 1997).

The high incidence of vasomotion in isolated irideal arterioles during the first three postnatal weeks coincided with the appearance and expansion of the perivascular, sympathetic neural plexus and the onset and maturation of nerve-mediated contractions (Sandow & Hill, 1997). This suggested that spontaneous contractions might result from spontaneous neurotransmitter release from the growing axons. However, spontaneous contractions were not affected by the sodium channel blocker tetrodotoxin, the adrenergic neurone-blocking drug guanethidine, the sensory neurotoxin capsaicin, or antagonists against muscarinic receptors, α-adrenoceptors, purinergic receptors or NPY Y1 receptors. The present results thus support the hypothesis that the vasomotion of irideal arterioles is myogenic, as it has been shown to be in other blood vessels (Hayashida et al. 1986; Bouskela & Grampp, 1992; Lee et al. 1994).

The tone of irideal vessels increased gradually with time in vitro and spontaneous contractions appeared routinely after 15-20 min incubation. A similar appearance of vasomotion with increase in tone has been described in isolated pressurized arterioles (Duling et al. 1981). The majority of drugs which caused an inhibition of spontaneous activity in the present study also caused a vasodilatation, suggesting that the initiation of spontaneous contractions may be related to the development of tension. Several observations, however, suggest that this was not the case. Reduction in the external concentration of calcium ions led to an abolition of vasomotion but no decrease in the resting tone. In similar fashion, there was no obvious loss of tone at the time when vasomotion ceased in irideal preparations. Cessation of vasomotion in isolated cannulated arterioles also occurred at a time when tonic contractions were stable for longer periods (Duling et al. 1981). Similarly, vasomotion in hamster cheek pouch arterioles in vivo was rapidly abolished by external conditions, such as increases in oxygen tension and changes in pH, without changes in vessel diameter (Bouskela & Grampp, 1992). Together, these results suggest that muscular tone, which may be dictated by the intracellular cytoplasmic calcium concentration, is not sufficient for the initiation of spontaneous activity and conversely, that the loss of spontaneous activity is not a result of the loss of tone of the arterioles.

Spontaneous rhythmical contractions of irideal arterioles were always preceded by large, slow depolarizations. Spontaneous contractions have also been observed to be associated with rhythmical depolarizations in the gastrointestinal tract (Tomita, 1981), urinary tract (Zhang & Lang, 1994; Hashitani et al. 1996), lymphatics (van Helden, 1993) and blood vessels (Segal & Beny, 1992; Gustafsson, 1993; von der Weid & Beny, 1993). In the present study, the amplitude of the spontaneous depolarizations was linearly correlated with the amplitude of the spontaneous contractions down to spontaneous depolarizations as small as 10 mV. These results are suggestive of a role for voltage-dependent channels. Contractile responses which depend on the influx of calcium through voltage-gated calcium channels are frequently found in depolarized preparations. This is the case for the myogenic response (Davis & Hill, 1999), where depolarization occurs in response to pressure and for certain vessels displaying vasomotion (Gokina et al. 1996). No significant changes in peak negative membrane potential of arterioles were, however, seen during development to correlate with the changing incidence of spontaneous contractions. Furthermore, no significant differences in peak negative membrane potential were found between arterioles which displayed vasomotion and those arterioles which were silent, suggesting that depolarization was not a prerequisite for spontaneous activity.

Caffeine and the calcium-ATPase inhibitors, thapsigargin and cyclopiazonic acid, rapidly abolished the spontaneous activity, although ryanodine was ineffective. These results suggest that the spontaneous contractions in the irideal arterioles result from the release of intracellular calcium, although ryanodine stores are not involved. While the spontaneous depolarizations in the urethra and lymphatics (van Helden, 1993; Hashitani et al. 1996) similarly resulted from the release of intracellular calcium, the rhythmical contractions were dependent on calcium influx through voltage-dependent calcium channels. Spontaneous contractions in irideal arterioles also depended ultimately on extracellular calcium, since they were abolished by the non- selective calcium channel antagonist, cadmium, as well as by reductions in the concentration of calcium in the external solution. Calcium influx, however, was not blocked by antagonists against L-type voltage-dependent calcium channels. Furthermore, in a previous study in the same vessels, depolarizations of around 30 mV were required before a contraction was initiated (Hill & Gould, 1997), while spontaneous depolarizations as small as 10 mV were associated with contractions in the present study. Taken together, these results suggest that the positive correlation between the amplitude of the depolarizations and the amplitude of the contractions arises due to their simultaneous activation by the same stimulus, rather than their sequential activation. We propose this stimulus to be a rise in intracellular calcium, for which the depolarization is more sensitive than the contraction. It is interesting that nerve stimulation of these arterioles produces a large, slow depolarization which precedes the contraction and results from the opening of calcium-dependent chloride channels (Gould & Hill, 1996).

While spontaneous rhythmical depolarizations and contractions did not depend on release of neurotransmitters from sympathetic or sensory nerves, stimulation of sensory nerves did lead to an inhibition of spontaneous activity. Release of calcitonin gene-related peptide from sensory nerves in this preparation has been shown to activate receptors which are coupled to both cAMP and nitric oxide pathways (Hill & Gould, 1997). The inhibition of spontaneous activity in the present study was not solely due to the receptor-mediated release of nitric oxide from the endothelium since incubation with L-NAME did not prevent the inhibition. On the other hand, increases in cAMP following activation of adenylate cyclase by forskolin, did abolish the spontaneous depolarizations and contractions. This observation coupled with our preliminary results that the inhibition of spontaneous activity following nerve stimulation is largely prevented by inhibition of adenylate cyclase suggests that increases in cAMP are important in this phenomenon.

Incubation in L-NAME alone resulted in an increase in the frequency of spontaneous depolarizations and contractions. Similar increases in frequency of vasomotion in hamster skeletal muscle microcirculation in vivo and in lymphatics in vitro were obtained following administration of inhibitors of nitric oxide synthesis (Bertuglia et al. 1994; von der Weid et al. 1996). These results suggest that vasomotion can be modulated negatively by endogenous release of nitric oxide from the endothelium. These effects differ from those described in small mesenteric arteries where endothelial release of nitric oxide and stimulation of smooth muscle cGMP is essential for agonist-induced vasomotion (Gustafsson, 1993). In the present study, there was no significant effect of L-NAME on the peak negative membrane potential suggesting that changes in the frequency of spontaneous contractions are not dependent on changes in membrane potential. This is further supported by the observations that α2-adrenoceptor agonists and the KATP channel antagonist glibenclamide, both cause depolarization (Hill & Gould, 1997), but only the former produces a change in the frequency of vasomotion (C. E. Hill, unpublished results).

Since some effects of cyclopiazonic acid have been suggested to occur through the stimulated release of calcium in the endothelium and subsequent activation of nitric oxide synthase (Huang & Cheung, 1997), the effect of cyclopiazonic acid was also tested after preincubation in the nitric oxide synthase inhibitor, L-NAME. While spontaneous contractions were inhibited in these experiments, the nerve-mediated response was unaffected, although it was abolished by the calcium-ATPase inhibitor, thapsigargin. A similar differential effect on the two types of contraction was seen in the present experiments after treatment with forskolin. Spontaneous contractions were abolished, but nerve-mediated contractions remained, when the indirect effects of cAMP on nitric oxide production were prevented (Gould et al. 1995; Fig. 5, present study). These results suggest that there may be multiple stores of intracellular calcium which are differentially sensitive to cyclopiazonic acid and forskolin. Furthermore, these different stores may be separately responsible for the spontaneous and receptor activated contractions.

Spontaneous depolarizations and contractions were rapidly abolished with the putative gap junction uncoupling agent, 18α-glycyrrhetinic acid (Davidson et al. 1986). The time course of the effect was very similar to that seen during the abolition of electrical coupling with 18β-glycyrrhetinic acid in guinea-pig mesenteric arterioles (Yamamoto et al. 1998), thus implicating gap junctions in the co-ordination of the spontaneous events. Considered together, the results of the present study suggest that the rhythmical contractions of irideal arterioles are independent of voltage-mediated events, but rather result from the co-ordination through gap junctions of cyclical fluctuations in calcium released from intracellular stores. These stores may differ from those accessed by receptor-mediated events. It seems unlikely that specific pacemaker cells initiate the oscillations, since preliminary studies on short segments of various lengths have found no evidence for changes in the appearance or frequency of vasomotion over that seen in intact arterioles (G. D. S. Hirst & C. E. Hill, unpublished observations). Furthermore, ultrastructural studies on these vessels have failed to find cells other than smooth muscle cells in the media (S. L. Sandow, personal communication). Vasomotion in irideal arterioles can be modulated by the action of sensory nerves and increases in cAMP, by the release of nitric oxide from the endothelium and presumably by other vasodilatory agents which activate these pathways. We hypothesize that gap junctions in these vessels co-ordinate calcium oscillations and consequent contractions by providing a pathway for chemical communication between adjacent smooth muscle cells, rather than for the transfer of electrotonic potentials.

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