the influence of the endothelium on the actions of acetylcholine in the isolated vascular ring preparation.

For your answers do not do a full report; simply answer the questions as numbered below (a) €“ (i).

Work that is not clear or not presented in a tidy, logical, concise way will lose marks.

Protocol
(1) Select arterial ring with endothelium intact.
(2) Expose the tissue to 300 nM phenylephrine €“ a concentration designed to give a near-maximal contraction. Record the tension induced.
(3) With the tissue now contracted, construct a complete cumulative dose response curve for acetylcholine, recording the reduction in tension at each concentration of ACh. You will need to experiment with different concentrations in order to obtain a complete dose-response curve.
(4) Discard the preparation, and select as your new tissue an arterial ring with endothelium removed.
(5) Repeat (2) and (3).
(6) Discard the tissue and repeat steps (1) and (2).
(7) AID 10??M L-NMMA.
(8) Now repeat step (3).
Results (steps 1-5 only)
(a) Record in a single table your Emax (i.e., maximal tension) values for phenylephrine
(0 nM ACh), and your corresponding responses to different concentrations of ACh in each tissue. (15 marks).
(b) Plot on one semi-log graph the log concentration/response curves for acetylcholine in the two preparations. Relaxant responses (y-axis) should be expressed as % relaxation (0-100% relaxation) of the initial contraction produced by phenylephrine. (15 marks).
(c) State your estimates of the IC50 values for the effects of acetylcholine in both types of arterial ring. (5 marks).

Results (Steps 6-8)
d) Plot on one semi-log graph the log concentration/response curves for acetylcholine in the presence and absence of L-NMMA. (15 marks).
e) Estimate the IC50 values for ACh in each case. (5 marks).

Questions
f) On what receptor type is phenylephrine mediating its response? (5 marks)
g) What receptor type is ACh binding to, to mediate its response? (5 marks)
h) Why is there such a pronounced difference between the two tissue preparations, in response to ACh? (Max: 100 words). (25 marks).
i) What is the effect of L-NMMA in this experiment, and why? (10 marks).
Reference:
P M Vanhoutte and T Scott-Burden (1994). The endothelium in health and disease. Tex Heart Inst J. 1994; 21(1): 62€“67.