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It has been observed in the past, that there was a progressive fall in responsiveness of vascular smooth muscle, such as in the rat tail artery, with time or with consecutive periods of activation by agonists, and this observation has apparently been recognized by many investigators (Medgett and Langer, 1986; Spedding, 1985; Su et al, 1984). But the lack of information on the possible causes of the initial decline between the first and the subsequent responses, has led to the neglect of the first concentration – response curve; the second or even the third being taken as the control curve for the analysis of test drugs (Medgett and Langer, 1986; Aoki and Asano, 1986).

First curves are discarded because a significant difference usually exists between the first and the subsequent curves, thereby creating problems for analysis of the data. The basis of this difference is unclear and it is reasonable to assume that the initial state is as likely to reflect the physiological properties of the tissue as does the subsequent desensitization condition: both cannot. An earlier study (McGrath et al, 1987b) showed that this desensitization can be reversed by leaving long (>2 hour) intervals between activations, and is thus not an inevitable deterioration with time.

We have now studied some of the experimental factors, which influence the initial and subsequent sensitivity to noradrenaline, and how this is influenced by the extracellular free calcium concentration. Strategies for avoiding desensitization are examined and the role of dihydropyridine-sensitive calcium channels is investigated using the calcium channel antagonist nifedipine and the calcium channel agonist Bay K 8644 (Schramm et al, 1983; Schramm et al, 1985). A preliminary communication of some of the results of this study carried out at the University of Glasgow, has been published (Ugwu et al, 1987).

METHODS

Preparation of the tissue for recording perfusion pressure: 1-2cm lengths of the proximal or distal segment of the rat tail artery were prepared for recording the perfusion pressure in vitro. Male Wistar rats (300 to 350 gm) were killed by a blow on the head and exsanguinations. The ventral tail artery was rapidly removed and placed in aerated ‘calcium-unbuffered’ modified Krebs’ bicarbonate solution. The vessel was cannulated at the proximal end and subsequently mounted in a 5ml jacketed organ bath. It was perfused with, and bathed in, saline of similar composition at 37⁰C.

Unbuffered saline: The ‘calcium-unbuffered, saline solution was made up of the following composition (in millimolar concentration):- NaCl, 119; NaHCO³, 24.8; KH₂PO₄,1.2; MgSo₄. 7H20, 1.2; KCl, 4.7; CaCl₂, 2.5; glucose, 11.1; cocaine, 4 m M; Propranolon, 1m; and Na₂EDTA (ethylene-diaminetetra-acetic acid disodium salt), 23mM.

Buffered saline: The ‘calcium-buffered’ saline solution has the following composition (millimolar unless otherwise specified):- EGTA (ethylene glycol bis- {B-aminoethyl ether} N,N,N’ – tetraacetic acid), 2.5 (i.e. : 0.9g^1-1) NTA (nitriol-triacetic acid, i.e. N,N-bis (carboxymethyl) glycerine, free acid); 2.5; NaCl, 111.5; NaOH,7.5; NaHCO₃,24.8; KH₃PO⁴, 1.2; MgSO₄. 7H₂O, 1.2; KCl, 4.7; CaC₂ was varied from 4.69 (for calcium buffer 1) to 2.35 (for calcium buffer 6); glucose 11.1; cocaine, 4 m M; Propanolol, 1mM;and EDTA (ethylyenediaminetetra-acetic acid disodium salt), 23 m M. The composition of the series of buffered salines is shown in Table 1. Another saline solution used in some experiments (examining the Ca²⁺) dependence of the contraction induced by depolarization) had high potassium chloride, low phosphate (which allowed the use of {Ca²⁺}o ³ 5mM without precipitation), and had identical composition to the one described above with the following exceptions:- NaCl, 24; KCl, 100; KH₂PO₄, 0.1.

Each saline was bubbled with a gas containing 95% and 5% C0₂, giving a partial pressure for oxygen of 615mmHg and aa pH of 7.2 to 7.4. In one series of experi6ments (see figure 6) the oxygen tension was varied by substituting N₂ for 0₂, to give 16% and 4% 0₂ as well as 95%: after such changes, 15 minute equilibration was allowed before introducing drugs or further altering the composition of the saline

Perfusion: The preparations were tested for leakage and those which were set up for perfusion. The arterial segments were mounted in the bath vertically with the cannulated proximal end of each tissue uppermost. The free distal end opened into the solution. The lumen was perfused from a gassed reservoir (37⁰C) at a constant rate of 2-3ml/min with a pulsate flow pump (Watson-Marlow peristaltic cassette pump, 501U with 501M multi-channel pump head) and the perfusion pressure was recorded. This rate of flow was shown in preliminary experiments to be adequate for recording the optimal vasoconstrictor responses to noradrenaline (NA) or to high concentration of potassium chloride (KCl).

The vasoconstrictor responses were measured as an increase in the peaks of the pulsatile perfusion pressure at constant average flow, using an Elcomatic EM751 pressure transducer and Devices recorder. The perfusatee passing through the artery via the cannular mixed freely with the identical saline solution in the organ that bathed the adventitial surface.

General Experiment Protocol: A standard stabilization period of 90 to 120mins in activator-free ‘Ca unbuffered’ solution was allowed before any responses were obtained. The protocol involved changing the perfusing solution by briefly stopping perfusion and switching it to new solution containing the required concentration of Ca²⁺, NA or KCl. The bathing solution in the organ bath was replaced simultaneously with the arrival of the new perfusate. Consequently both surfaces of the tissues were always exposed to an identical medium during the experiments. (In practice, the responses to noradrenaline were not larger when it was presented to both surfaces than to the initima alone, using double cannulation {data not shown} but in order to achieve equlibrium conditions the preceeding protocol was adopted). Standard exposure of the tissues to NA or KCl with either cumulative or non-cumulative protocols was for 5 mins during which time the maximum response to that concentration of the activator was obtained. In some experiments, as indicated in the text, tissues were exposed to initial ‘priming’ concentrations of noradrenaline in various buffers for 5 min before starting the main protocol.

In all experiments involving responses to activators, EGTA and NTA were included (2.5mM each) so that any toxic effect would be constant throughout, and unless a particular buffers is specified total CaCl₂ was adjusted to keep [Ca²⁺] free at 2.5mM. In all the experiments involving NA-induced contractions, cocaine (4 m M), propranolol (1 m M) and EDTA (23 m M) were present.

Noradrenaline concentration/response curves: After equlibration for 15min in the appropriate saline buffer (starting at the lowest concentration of calcium and working up), concentration/responses curves to noradrenaline were constructed non-cumulatively, with 5min contact and 10min wash, starting at the lowest concentration, 30nM, and proceeding in half log unit step to 30 m M. 15min intervals were left between curves in different calcium buffers, giving a routine cycle time for noradrenaline concentration/responses curves of 110min.

In these experiments the tissues were initially exposed to 3 m M noradrenaline for 5min as part of a stabilization procedure, 15min before starting the first concentration/responses curve. In retrospect, this is likely to have caused some desensitization and this will be considered in the discussion.

Calcium concentration/responses curves: Concentration/response curves to Ca²⁺ (CCRC) were constructed by changing to the lowest [Ca²⁺] (buffer 6; see Table 1) for 15min, adding the activator (NA or KCl) then, starting 5mins later, changing stepwise to higher [Ca²⁺] at 5mins interval starting from buffer 6 [Ca²⁺]o =10^-6 M] through a series of buffers referred to as buffers 5 to 1 which took the [Ca²⁺] to 300 m M. In some experiments, further increments in [Ca²⁺] free of 5.32mM or at times to 10. 32mM. Details of the [Ca²⁺] in the various buffers are outlined in Table 1.

The concentration of NA (3mM) or KCl (100mM) was kept constant while changing [Ca²⁺]. At the end of the construction of the CRCS, the perfusion was stopped and the bathing and perfusing solutions were replaced by activator-free buffer 6 solution and allowed to re-equilibrate (to baseline response) before constructing another CCRC. Preliminary experiments showed that washing and “resting” in buffer 6 minimized desensitation, i.e. it was worse if [Ca²⁺] was higher. Intervals of 15min were allowed between curves in the initial protocol but this was varied in other experiments as noted in the text. A total time of 45minutes was taken to complete the construction of each control curve, giving a routine cycle of 1 curve per hour.

Table 1 Ca²⁺ concentrations in the buffers used

Buffer	[Ca²⁺] free	[Ca²⁺] total
	(M)	(mM)
6	1 x 10^-6	2.35
5	3 x 10^-6	2.47
4	1 x 10^-5	2.69
3	3 x 10^-5	3.12
2	3 x 10^-4	3.90
1	3 x 10^-4	4.69
‘half Ca²⁺	1.25 x 10^-3	[Buff 1] + 1.57
‘normal’ Ca²⁺	2.50 x 10^-3	[Buff 1] + 2.83
‘double’ Ca²⁺	5.00 x 10^-3	[Buff 1] +5.32
‘qudruple’ Ca²⁺	10.00x10^-3	[Buff 1] + 10.32

Drugs and chemicals

The following substances were used:- (-)-noradrenaline bitartrate salt (sigma), Bay K 8644 (Bayer), nifedipine (Bayer), Cocaine HCl (McCarthys), DL-Propranolol HCl (I.C.I.), E.D.T.A. (B. D. H.), E.G.T.A. (ethylene glycol bis-{B-aminoethyl ether} N,N,N’-tetraacetic acid (Sigma), N.T.A. (nitrilo-triacetic acid, i.e., N,N-bis {carboxymethyl} glycine, free acid) (Sigma). Stock solutions of drugs were dissolve in distilled water, v/v and diluted in the appropriate saline.

Expression of data: All results have been expressed, or represented on graphs, as the mean ± S.E.M. Statistical analysis was performed using Student’s ‘t’ test for paired or unpaired data, as appropriate and the 0.05 level of probability was regarded as significant.

Before sensitization occurred the preparations characteristically showed a ‘peaked’ concentration/response curve to agonists, or to calcium concentration at a fixed concentration of agonist. Both ‘sensitivity’ and maxima subsequently declined, if steps were not taken to avoid desensitization and, in many cases, a ‘maximum’ was not obtained within the possible range of soluble calcium. This raises problems in quantifying calcium sensitivity, since there was often no maximum, within a given curve, on which to base the 50% response for calculation of a pD₂ value. Furthermore, after desensitization, the curves sometimes did not attain 50% of the maximum from the first curve. For simplicity we have expressed sensitivity in each curve in relation to the calcium concentration which allows a response of 30% of the maximum obtained in the first curve constructed, i.e. ‘EC₃₀’ = concentration of calcium allowing a response of 30% of initial maximum, obtained by graphical interpolation. This corrects for inter-tissue variations in the absolute size of responses but makes no assumptions about the history of the preparation after the first curve. Thus as desensitization proceeds, the EC₃₀ steadily increases and –log EC₃₀ falls. For each group of experiments sensitivity is expressed as the arithmetic mean of the –log Ec₃₀ values ± S.E. mean.

RESULTS

Concentration/response curves for NA in varying (Ca²⁺): In the calcium-buffered saline, in which a range of low concentration of (ca²⁺) could be employed, responses to NA increased from 1 mM (buffer 6) to 0.3mM (buffer 1) (Fig. 1a) (see Table 1 for the concentration of free calcium concentrations in the buffers).

From this data Ca CRCs can be constructed for each NA concentration, (0.1, 0.3, 1.0 and 3mM), the responses increasing with increasing Ca²⁺ CRC shown in figure 3. the sensitivity to NA calculated in this way approached that found in the ‘standard’ [Ca²⁺] of 2.5mM in virgin tissue, but not that found after the ‘stabilization procedure’ (Fig. 1c).

When the pS2 values (-log EC₅₀) were calculated for the NA concentration/response curves in figure 1a, a decline in sensitivity to noradrenaline was found as [Ca²⁺]o declined (fig. 1c).

Prolonged responses to NA in [Ca²⁺] free = 2.5mM: Since sensitivity to [Ca²⁺] was to be assessed, in subsequent experiments, by cumulative CRCs lasting 45mins each, a total of six consecutive 45mins long exposures to NA 3m M were produces in 2.5mM Ca²⁺ after the 3^rd. when this was compared with the NA 3 responses at m M 2.5mM (Ca²⁺) obtained in Ca CRCs (see below), the magnitudes of the pressor were similar (FIG 2.). Having established that prolonged responses show desentisation at a constant high level of (ca²⁺) some factors influencing Ca²⁺ sensitivity during sensitization were then studied.

Ca²⁺ sensitivity of contractile responses to NA and K⁺: Arteries were exposed to a concentration of 3mM NA or 100mM K⁺ in the presence of a low concentration of free calcium ions in solution (buffer 6). Cumulative increases of [Ca²⁺] free up to 5.32mM (twice the calcium concentration commonly employed], elicited concentration-dependent contractions.

Six such Ca²⁺ concentration response curves (CCRC) were obtained at 45 minutes intervals. In such a series, the sensitivity of the tissues to [Ca²⁺] free steadily declined. The maximum responses, the-log (EC³⁰) and the – log (EC⁵⁰) of the first curves were statistically significantly greater than in the second or subsequent curves. For NA there was a progressive shift which slowed after the 1^st CCRC. However, for KCl-induced responses the second and the subsequent curves were not statistically different from each other in any of the above parameters.

(i). Noradrenaline

For NA (3mM), the first CCRC lay further left than the subsequent curves (by 1.13 +s 0.20 log units, n = 6, at the level of –log EC³⁰ values) compared with the 2^nd. This represents the distance between the means of the individual pairs of EC³⁰s. This first CCEC declined after reaching a peak. It peaked at [Ca²⁺] free = 0.3 m M to 1.25mM with 30% (i.e. approximately 50mmHg) of its maximum attained by 30 m M). The second and subsequent CCRCs showed a more sigmoid correlation of response with log [Ca²⁺] and responses had not attained a true maximum even at 5mM. The second curve had reached 50mmHg (which is approximately 50% of its “maximum”) by 300 m M; and the third CCRC by about 600 m M, with the rightward shift slowing down thereafter. For example, the 6^th CCRC attained 50mmHg by [Ca²⁺] = 1mM (Fig. 3).

The peaked first curve makes correction of responses to ringer-tissue variability difficult. Furthermore, comparison of Ca²⁺ sensitivity in different conditions, even in a single tissue, is not straightforward when the “slope” and maximum are changing. We have expressed all pressor responses as a percentage of the 1^st maximum to the particular activator, whether this was obtained in a CCRC or during priming.

These 1^st maxima were not significantly different between series with the exception of those in the presence of nifedipine. Thus we have compensated only for tissue variability in the height of responses. Ca²⁺ sensitivity was expressed as the concentration producing 30% of this 1^st maximum (EC₃₀) (interpolated as –log EC₃₀). Thus, when the maximum changes, this –log EC₃₀ value no longer represents a true –log EC₃₀ for that curve, but a slightly lower value, exaggerating the extent of desensitization. However even expressed as a percentage of the maximum within each curve, statistically significant desensitization still occurs (data not shown).

The rightward shift (decline) in the sensitivity of tissue to calcium (represented by the EC₃₀ values) (Fig. 3) continued with subsequent curves after the second, but to a smaller degree. This rightward shift of the EC₃₀values, expressed as the –log EC₃₀, was used as an index of the fall in sensitivity (Fig. 4a).

(i).Potassium Chloride: High potassium chloride (KCl, 100mM), also produced a peaked first curve which was not repeated in the 2^nd or subsequent curves and was, in this respect, similar to NA. However, the [free Ca²⁺] required for any given response (at 2.5mM Ca²⁺) with KCl was approximately 10 times higher tha with NA (Fig. 4b). The [free Ca²⁺] for a 50mMHg response changes from just above 100 m M in the first curve to 1.25mM in the second curve – a rightward log shift of 0.91 + 0.15 at the level of EC₃₀ values, i.e. not significantly different from the situation with NA (1.13 ± 0.2).

Effects of the range of [Ca²⁺] free used to construct the CRC: The highest concentration of [Ca²⁺], to which the NA-activated tissue was exposed, affected Ca²⁺ -sensitivity. At the first determination of Ca²⁺ -sensitivity, the maximum concentration usually occurred by 300 m M Ca²⁺. When the tissue was exposed to a maximum of 300 m M Ca²⁺ (buffer 1), desensitization of subsequent CRC’s was less than when [Ca²⁺]o was taken up to 5mM. Another factor enters these later experiments since the tissues were exposed to NA for a shorter time (30min), which might have accounted for less desensitization. However, desensitization was still reduced even when the tissues were left contracted in 300 m M Ca²⁺ for longer so that the total time for constructing each CCRC was 45min, i.e. the same as for the controls which were exposed to a maximum 5mM Ca²⁺ (Fig. 5a).

Effect of a “priming” contraction to NA in different levels of [free Ca²⁺] before constructing CCRCs:

(i)“Priming” in Buffer 1 (300 m M Ca²⁺): If the tissue were exposed for 5min to NA (3 m M) in Buffer 1 (“primed”) before constructing the first CCRC (up to 5mM Ca²⁺), desensitization was partially arrested (Fig. 5b). Therefore either priming in 300 m M Ca²⁺ or taking CRCs to a ‘maximum’ [Ca²⁺] of 300 m M reduced desensitization. Combining the two was even more effective: the EC₃₀ for the 6^th CRC was not significantly different from the first (Fig. 5b).

(ii). “Priming” in 2.5mM Ca²⁺: Priming in 2.5mM Ca²⁺ accelerated desensitization for the 1^st two curves although there was some recovery thereafter. This shifted the first curve to the position of the usual second curve. The second curve was shifted to the position of the usual sixth curve (Fig. 5c). Thus while this procedure produced a degree of stability, it did so by making the tissues insensitive to Ca²⁺ by approximately one log unit.

Effect of Bay K 8644 and Nifedipine on the CCRC and on desensitsation

(i). Bay K 8644: Bay K 8644 (0.1 m M) on its own, unlike the activators NA or KCl, did not produce any change in the baseline, when it was added at any point within the range of [Ca²⁺] free used. However, it potentiated the first CCRC with NA or KCl (Fig. 4a&b).

Although Bay K 8644 increased the sensitivity to CA²⁺, desensitization still occurred. However, the EC₃₀ in each CCRC indicated greater sensitivity than in the equivalent Bay K 8644-free time control (Fig. 4).

The use of EC₃₀ based on initial maximum was not significantly altered by Bay K 8644 (0.1 m M). The calcium threshold value for a pressor response was alos less when Bay K 8644 was present in the saline. In general, for a given CCRC (1^st, 2^nd etc) the effect of Bay K 8644 was a parallel displacement to the left, whether the responses were induced by NA or KCl in calcium-buffered saline. Due to the progress of desensitization, addition of Bay K 8644 during construction of a series of curves needs to take into account the time effect. However its potentiating effect can still be clearly seen as is demonstrated in figure 6.

This figure was taken from a study of the effects of oxygen tension on calcium sensitivity, which basically showed that oxygen tension over the range studied had little effect on its own, but that Bay K 8644 potentiated at all oxygen tensions compared with time controls. This was confirmed by repeating the entire protocol at each of these oxygen levels

(ii). Nifedipine

Nifedipine (0.1 m M), attenuated the calcium-dependent contractions of the rat tail artery to noradrenaline and KCl, without altering the baseline. For contractions to noradrenaline, nifedipine decreased sensitivity to Ca²⁺ at the first CCRC compared with untreated controls but thereafter CRC’s were not significantly different from their time controls. In contrast, if nifedipine was absent for the first 3 or 6 CRC’s, when it was subsequently added it tended to increase the rate of fall in Ca²⁺ -sensitivity at the next test, though the effect was small (Fig. 7 and fig 4a).

The inhibition of responses to KCl was more clear-cut. Even after desensitization (6 CCRCs) nifedipine caused a further reduction in sensitivity to Ca²⁺ (fig. 4b). Basing Ca²⁺ sensitivity on EC_30S calculated on the initial maximum is not straightforward with nifedipine since it made the 1^st maximum significantly smaller. This has the effect of overestimating sensitivity to [Ca²⁺] after nifedipine when comparing with other situations. This can be taken into account by

(i) Calculating EC₃₀ using the actual maximum in each individual curve, or
(ii) Estimating the [Ca²⁺] which produces a fixed absolute response.

In either case, compared with time controls, nifedipine (0.1m M) led to a significant decrease (p<0.05) in sensitivity of the 1^st curve but subsequently this was similar to the time controls (data not shown).

DISCUSSION

The sensitivity to calcium of noradrenaline-induced vasoconstriction of rat tail artery starts at a high value and declines as the experiment progresses. This causes ‘desensitization’ both to calcium, and when noradrenaline concentration response curves are analysed, to noradrenaline (McGrath et al, 1987a: 1987b). An initial deliberate desensitization procedure leaves a stable but, by definition, less sensitive preparation.

To elucidate the mechanism underlying desensitization, we have examined some factors which influence it, concentrating on the sensitivity to [Ca²⁺]o since this shows up desensitization even when the response in 2.5mM Ca²⁺ is little altered. It was clear even when constructing the first CCRC that high [Ca²⁺] was deleterious to the preparation.

Modifying the experimental protocol showed that the highest calcium concentration to which the tissue was exposed affected Ca²⁺ -sensitivity. The higher the [Ca²⁺]o, the greater the desensitization. This suggests that some form of “Ca overload” may be responsible for the deterioration of responses in this smooth muscle as it does in cardiac muscle (Allen et al, 1985). It has been propose that an internal binding site for calcium on the cell membrane regulates the rate of desensitization (Nastuk and Parsons, 1970; Debassio et al, 1976).

An accumulation of excess free intracellular calcium, in “ca overload”, may contribute to desensitization of this tissue via such an internal binding site. This seems to be the case also in guinea-pig ileum where excess calcium accelerates desensitization (Magaribuchi et al, 1973) the different degrees of desensitization produces by priming in different concentrations of ca²⁺ show that priming can produce some stability of subsequent responses but that the remaining level of sensitivity will depend on [Ca²⁺] both during priming and in the subsequent tests. Therefore priming accelerates desensitization but leaves the tissue relatively insensitive.

This stabilization explains why many workers initially activate their preparation several times until the responses are reproducible (Su et al, 1984; Aoki and Asano, 1986). Clearly, avoidance of high (Ca²⁺ ) during priming as well as during subsequent parts of the protocol lead to stable preparations which are significantly less desensitized, since cross-bridge power calculated on the basis that all cross-bridges contribute equally to power production (Niggli, 1999) it is interesting to note that priming in 300mM Ca²⁺ (buffer 1) lead to desensitization. Possibly, on it 1^st activation, this lead to Ca overload; but if (Ca²⁺ ) is not high the 1^st time the tissue is activated, then Ca overload is lessened. Carrying on the hypothesis, second activation produces fewer channel openings. Therefore, if priming is carried out in a protective, low calcium environment, subsequent high sensitivity is ensured, particularly if high calcium is still avoided. Nevertheless if original sensitivity is of interest then perhaps it is best to avoid activation by high (noradrenaline) or high (KCI) altogether. According to such a hypothesis, the site of an initial element is desensitization would lie between receptor activation and the part of the channel responsible for its opening. Some deficit induced here after first activation, e.g. depletion of second messenger substrate, would lead to fewer openings on subsequent activations. The main “damage” however, would be caused by excessive entry.

It was observed in an earlier study that a small concentration of NA (0.3mM), which is one-tenth of the concentration used here, led to less desensitization after the first curve and the subsequent curves stabilized at a higher level of sensitivity than with 3mM NA. This again suggests that extracellular (Ca²⁺) is not the only factor in desensitization. A further requirement is a sufficiently high stimulus from the activator, possibly by an increased opening of Ca²⁺ channels (either more channels open per unit time or longer open-time per channel).

Bay K 8644 increased Ca²⁺ -sensitivity but did not alter desensitization. This suggests that Bay 8644 sets up a new equlibrium position for tissue responses by allowing further opening of Ca²⁺ channels by the activating stimulus but that at the same time if allows the accumulation of Ca²⁺, perhaps even enhancing it. This would explain the combination of desensitization within each preparation coupled with potentation of responses relative to time controls.

Nifedipine decreased Ca²⁺ -sensitivity. Interpretation of the effects of nifedipine is not straightforward. Clearly, nifedipine prevented the characteristic 1^st responses if given before any activation had occurred. Subsequent responses, however, were no different from time controls (expressed as –log EC₃₀). This suggests that the initial high sensitivity is produced by a high functional effectiveness of dihydropyridine-sensitive channels and that desensitization in large part consists of the loss of their function.

On its own, the observation that the second CCRC in the presence of nifedipine similar to its time control suggests that after the 1^st curve, i.e., in desensitized tissue the main part, if not all, of the response involves Ca²⁺ influx through dihydropyridine-insensitive channels. The acute effect of nifedipine, given after partial desensitization, suggests that in desentised tissue which have not been exposed to nifedipine, some dihydropyridine-sensitive channels remain functional but their contribution to the response is relatively trivial. Thus, dhydropyridine-sensitive channels are significantly involved in the initial response to NA but this element is largely lost after desensitization.

In order to study the calcium sensitivity of noradrenaline-induced vvasoconstriction, it was necessary to select an appropriate concentration of noradrenaline, which could produce well maintained and reproducible responses, and preferably, be sub-maximal. Since desensitization of the tissue combined with normal inter-tissue variation can produce substantial shifts in the noradrenaline pD₂value across two orders of magnitude, we selected a concentration of 3mM which is maximal in non-desensitized tissue and still approximately 80-90% of maximum in “stabilized” desensitized tissue (data not shown). Since changing the calcium level might be expected to change the efficiency of receptor-contraction coupling, it is possible that the receptor reserve is altered. The noradrenaline concentration/response curves in different calcium buffers are a check on this; they show that there is a decline is the noradrenaline pD₂ value as calcium concentration declines but that, over the calcium concentration range in which the responses are accurately measurable, this effect is not substantial. Furthermore, since the protocol employed a ‘priming’ exposure to noradrenaline in 2.5mM calcium, it is likely that the sensitivity to noradrenaline shown in the lowest one or two levels of calcium is a slight underestimate. It seems, therefore, that in this tissue and with the receptor system involved, receptor reserve is not greatly influenced within the range of extracellular calcium levels which can sustain responses.

With this particular protocol, responses to norarenaline were highly sensitive to praxosin (results not shown) and are therefore interpreted as being primarily due to a₁-adrenoceptors, although other preparations of the vasculature of the rat tail can show a₂-adrenoceptor-mediated vasoconstriction (Treager et al, 1998; Maurghan and Vigoreaux, 1999): since the _B – adrenergic stimulation can prolong the open time of the L-type Ca²⁺ channels (Templeton et al, 1989).

In conclusion, we have established several factors which influence desensitization in the smooth muscle of rat tail artery. Our results are consistent with a desensitization of Ca²⁺ (2.5 or 5mM) is present during, or subsequent responses. This can be reversed spontaneously by leaving long intervals between tests (6) or can be avoided restricting Ca²⁺ to < 300mM during activation. Some stabilization of sensitivity can be achieved by a ‘ priming’ concentration in Ca²⁺ (300mM or 2.5mM) but this does seem that the initial high sensitivity to Ca²⁺ is a state which is “normal’ for this tissue in vitro unless it is exposed to the vigorous insult of a prolonged concentration by a non-physiological concentration of NA at a high [Ca²⁺].

ACKNOWLEDGMENTS

We the authors are grateful to the Commonwealth Scholarship Commission in London for the Commonwealth Scholarship Award to A. C. U, with which the study was carried out.

REFERENCES