Your slope is wrong, plus I made numerous other improvements. See this:
clc; % Clear the command window.
close all; % Close all figures (except those of imtool.)
imtool close all; % Close all imtool figures if you have the Image Processing Toolbox.
clear; % Erase all existing variables. Or clearvars if you want.
workspace; % Make sure the workspace panel is showing.
format long g;
format compact;
grayImage = imread('pout.tif');
imshow(grayImage);
% Enlarge figure to full screen.
set(gcf, 'units','normalized','outerposition',[0 0 1 1]);
% Give a name to the title bar.
set(gcf,'name','Demo by ImageAnalyst','numbertitle','off')
[rows, columns] = size(grayImage)
message = sprintf('Click and drag the endpoints of the line\nand double-click the double arrows to finish.');
uiwait(helpdlg(message));
h = imline;
lineEndPoints = wait(h)
delete h;
x1 = lineEndPoints(1,1);
y1 = lineEndPoints(1,2);
x2 = lineEndPoints(2,1);
y2 = lineEndPoints(2,2);
hold on
plot([x1,x2],[y1,y2], 'r-', 'LineWidth',2); % Make line.
plot([x1,x2],[y1,y2], 'yo', 'LineWidth',2,'MarkerSize', 12); % Make circles.
message = sprintf('Click a third point.');
uiwait(helpdlg(message));
[x3, y3] = ginput(1);
plot(x3, y3,'yo','MarkerSize', 12);
% Calculate the slope.
slope = (y2 - y1)/ (x2 - x1)
% Draw second line.
x4=0;
x5=rows;
y4 = slope * (x4 - x3) + y3
y5 = slope * (x5 - x3) + y3
plot([x4,x5 ],[y4,y5],'r-','LineWidth', 2);
